Cancer associated antigens, sga-56m and sga-56mv, and uses thereof

ABSTRACT

The present invention relates to nucleic and amino acid sequences of SGA-56M and variants thereof. An exemplary variant of SGA-56M is SGA-56Mv. SGA-56M and SGA-56Mv are differentially expressed in cancer tissues and transformed cell lines. In particular, these genes are expressed at elevated levels in breast cancer and lung cancer tissues and cell lines. The present invention is also directed to methods for diagnosing a cancer in a subject, determining the prognosis of a cancer patient, and/or monitoring the efficacy of a treatment regimen for a cancer patient. The present invention further provides methods for screening and identifying modulators (e.g., antagonists or agonists) of SGA-56M and/or SGA-56Mv expression and/or activity. Antagonists identified using the methods of the invention are useful for the treatment of cancer patients. Also encompassed by the present invention are compositions including nucleic and/or amino acid sequences of SGA-56M and SGA-56Mv or SGA-56M/SGA-56Mv modulators and a pharmaceutically acceptable carrier. Such compositions may be used to advantage to treat a subject with a cancer to ameliorate the symptoms of the cancer or reduce the cancer cell burden in the subject. These compositions may also be utilized to prevent or delay the onset of a cancer in a subject exhibiting a predisposition to developing a cancer.

This application claims priority under 35 USC §119(e) from U.S.Provisional Application Ser. No. 60/410,048 filed 12 Sep. 2002, whichapplication is herein specifically incorporated by reference in itsentirety.

1. FIELD OF THE INVENTION

The invention relates generally to the field of cancer diagnosis,prognosis, treatment and prevention. More particularly, the presentinvention relates to methods of diagnosing, treating and preventingbreast cancer. Methods of using a nucleic acid and a protein,differentially expressed in tumor cells, and antibodies against theprotein, to treat, diagnose or prevent cancer, are provided for by thepresent invention. The instant invention provides compositionscomprising, and methods of using, products of a gene termed SGA-56M andvariants thereof, including SGA-56Mv. Such SGA-56M gene products includeSGA-56M proteins and nucleic acids and variants thereof, includingSGA-56Mv. Such gene products, as well as their binding partners andantagonists, can be used for the prevention, diagnosis, prognosis andtreatment of cancer.

2. BACKGROUND OF THE INVENTION

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastases). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia.

Pre-malignant abnormal cell growth is exemplified by hyperplasia,metaplasia, or most particularly, dysplasia (for review of such abnormalgrowth conditions, see Robbins & Angell, 1976, Basic Pathology, 2d Ed.,W.B. Saunders Co., Philadelphia, pp. 68-79) The neoplastic lesion mayevolve clonally and develop an increasing capacity for growth,metastasis, and heterogeneity, especially under conditions in which theneoplastic cells escape the host's immune surveillance (Roitt, I.,Brostoff, J. and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis,pps. 17.1-17.12).

The incidence of breast cancer, a leading cause of death in women, hasbeen gradually increasing in the United States over the last thirtyyears. Its cumulative risk is relatively high, 1 in 8 women, forexample, by age 85 in the United States. In fact, breast cancer is themost common cancer in women and the second most common cause of cancerdeath in the United States. In 1997, it was estimated that 181,000 newcases were reported in the U.S., and that 44,000 people would die ofbreast cancer (Parker et al., 1997, CA Cancer J. Clin. 47:5; Chu et al.,1996, J. Nat. Cancer Inst. 88:1571). While the mechanism oftumorigenesis for most breast carcinomas is largely unknown, there aregenetic factors that can predispose some women to developing breastcancer (Miki et al., 1994, Science 266:66). The discovery andcharacterization of BRCA1 and BRCA2 has expanded our knowledge ofgenetic factors that can contribute to familial breast cancer. Germ-linemutations within these two loci are associated with a 50 to 85% lifetimerisk of breast and/or ovarian cancer (Casey, 1997, Curr. Opin. Oncol. 9:88; Marcus et al., 1996, Cancer 77: 697). Sporadic tumors, however, orthose not known to be associated with a known germline mutation,constitute the majority of breast cancers. It is likely that other,non-genetic factors also have a significant effect on the etiology ofthe disease. Regardless of its origin, breast cancer morbidity andmortality increases significantly if it is not detected early in itsprogression. Thus, considerable effort has focused on the earlydetection of cellular transformation and tumor formation in breasttissue.

Only about 5% to 10% of breast cancers are associated with breast cancersusceptibility genes, BRCA1 and BRCA2. The cumulative lifetime risk ofbreast cancer for women who carry the mutant BRCA1 is predicted to beapproximately 92%, while the cumulative lifetime risk for thenon-carrier majority is estimated to be approximately 10%. BRCA1 is atumor suppressor gene that is involved in DNA repair and cell cyclecontrol, which are both important for the maintenance of genomicstability. More than 90% of all mutations reported so far result in apremature truncation of the protein product with abnormal or abolishedfunction. The histology of breast cancer in BRCA1 mutation carriersdiffers from that in sporadic cases, but mutation analysis is the onlyway to identify a carrier. Like BRCA1, BRCA2 is involved in thedevelopment of breast cancer and plays a role in DNA repair. However,unlike BRCA1, it is not involved in ovarian cancer.

Other genes have been linked to breast cancer, for example c-erb-2(HER2) and p53 (Beenken et al. 2001, Ann Surg. 233(5):630). Overexpression of c-erb-2 (HER2) and p53 have been correlated with poorprognosis (Rudolph et al. 2001, Hum. Pathol. 2(3):311), as has beenaberrant expression products of mdm2 (Lukas et al. 2001, Cancer Res.61(7):3212) and cyclin1 and p27 (Porter & Roberts, InternationalPublication WO98/33450, published Aug. 6, 1998).

A marker-based approach to tumor identification and characterizationpromises improved diagnostic and prognostic reliability. Typically, thediagnosis of breast cancer and other types of cancer requireshistopathological proof of the presence of the tumor. In addition todiagnosis, histopathological examinations also provide information aboutprognosis and selection of treatment regimens. Prognosis may also beestablished based upon clinical parameters such as tumor size, tumorgrade, the age of the patient, and lymph node metastasis.

In clinical practice, accurate diagnosis of various subtypes of canceris important because treatment options, prognosis, and the likelihood oftherapeutic response all vary broadly depending on the diagnosis.Accurate prognosis, or determination of distant metastasis-free survivalcould allow the oncologist to tailor the administration of adjuvantchemotherapy, with patients having poorer prognoses being given the mostaggressive treatment. Furthermore, accurate prediction of poor prognosiswould greatly impact clinical trials for new breast cancer therapies,because potential study patients could then be stratified according toprognosis. Trials could then be limited to patients having poorprognosis, in turn making it easier to discern if an experimentaltherapy is efficacious. To date, no set of satisfactory predictors forprognosis based on the clinical information alone has been identified.The detection of BRCA1 or BRCA2 mutations represents a step towards thedesign of improved therapeutics and therapeutic regimens for preventingand regulating the appearance of these tumors.

It would, therefore, be beneficial to provide specific methods andreagents for the diagnosis, staging, prognosis, monitoring and treatmentof cancer, including breast cancer, and to provide methods foridentifying individuals with a predisposition for the onset of breastcancer, and other types of cancer, and hence are appropriate subjectsfor preventive therapy.

3. SUMMARY OF THE INVENTION

Intensive and systematic evaluation of gene expression patterns isessential in understanding the physiological mechanisms associated withcellular transformation and metastasis associated with cancer. Severaltechniques that permit comparison of gene expression in normal andcancerous cells are known in the art Examples of these techniquesinclude: Serial Analysis of Gene Expression (SAGE) (Velculescu et al.,1995, Science 270:484); Restriction Enzyme Analysis of DifferentiallyExpressed Sequences (READS) (Prasher et al., 1999, Methods in Enzymology303:258); Amplified Fragment Length Polymorphism (AFLP) (Bachem et al.,1996, Plant Journal 9:745); Representational Difference Analysis (RDA)(Hubank et al., 1994, Nucleic Acid Research 22:(25):5640); differentialdisplay (Liang et al., 1992, Cancer Research 52(24):6966); andsuppression subtractive hybridization (SSH) (Diatchenko et al., 1996,Proc. Natl. Acad. Sci. USA 93:6025). As described herein, the presentinventors have used differential expression methods to identify andcharacterize the SGA-56M gene and variants thereof, including SGA-56Mv,as genes whose expression is associated with breast cancer and othertypes of cancer. This discovery by the present inventors has madepossible the use of SGA-56M and variants thereof, including SGA-56Mv,for the treatment, prevention and diagnosis of cancers, including butnot limited to breast cancer.

The present invention relates to the discovery that a gene, SGA-56M andvariants thereof, including SGA-56Mv, has an expression pattern that isup-regulated in cancer tissues and cell lines, e.g., breast cancertissues and cell lines. The invention relates to the use of said gene,gene products, and antagonists of said gene or gene products (SGA-56Mand variants thereof, including SGA-56Mv, cDNA, RNA, and/or protein) astargets for diagnosis, drug screening and therapies for cancer. Thepresent invention also relates to the use of said genes or gene productsor derivatives thereof as vaccines against cancer. In a preferredembodiment, the invention provides for methods of using the protein,SGA-56M and variants thereof, including SGA-56Mv, or nucleic acids thatencode said proteins for the treatment, prevention and diagnosis ofbreast cancer.

In particular, the methods of the present invention include usingnucleic acid molecules that encode the SGA-56M protein and variantsthereof, including SGA-56Mv, and recombinant DNA molecules, cloned genesor degenerate variants thereof, and in particular naturally occurringvariants that encode SGA-56M related gene products. The methods of thepresent invention additionally include using cloning vectors, includingexpression vectors, comprising the nucleic acid molecules encodingSGA-56M and variants thereof (e.g., SGA-56Mv), and hosts that comprisesuch nucleic acid molecules. The methods of the present invention alsoencompass the use of SGA-56M gene products and variants thereof,including SGA-56Mv, fusion proteins, and antibodies directed againstsuch SGA-56M gene products or conserved variants or fragments thereof.In one embodiment, a fragment or other derivative of an SGA-56M proteinis at least 10 amino acids long. In another embodiment, a fragment of anSGA-56M nucleic acid and variants thereof, including SGA-56Mv, nucleicacid or derivative thereof is at least 10 nucleotides long.

The nucleotide sequence of the cDNA of a human SGA-56M gene, andSGA-56Mv is provided. The nucleotide sequences of the SGA-56M ORF, andSGA-56Mv ORF in the SGA-56M, and SGA-56Mv genes, as well as the aminoacid sequences of the encoded gene products, are also provided. TheSGA-56M and SGA-56Mv genes were cloned by PCR. The SGA-56M transcriptencodes a protein of 802 amino acids and the SGA-56Mv transcript encodesa protein of 756 amino acids. The SGA-56Mv protein has an in-framedeletion of amino acids 234-280 of SGA-56M. In-frame start and stopsequences were observed by sequence analysis of the SGA-56M and SGA-56Mvgenes. The SGA-56M and SGA-56Mv transcripts were both detected atelevated levels in both breast cancer cell-lines and breast tumorisolates as compared to normal tissues. Elevated transcript levels ofSGA-56M and SGA-56Mv genes were also associated with lung cancer tissue.Elevated transcript levels were also detected in several other tumortypes and cancer cells as described in Section 6.

The present invention further relates to methods for the diagnosticevaluation and prognosis of cancer in a subject animal. Preferably thesubject is a mammal, more preferably the subject is a human. In apreferred embodiment the invention relates to methods for diagnosticevaluation and prognosis of breast cancer. For example, nucleic acidmolecules of the invention can be used as diagnostic hybridizationprobes or as primers for diagnostic PCR analysis for detection ofabnormal expression of SGA-56M and SGA-56Mv genes.

Antibodies or other specific binding partners to the SGA-56M andvariants thereof, including SGA-56Mv proteins, of the invention can beused in a diagnostic test to detect the presence of the SGA-56M orSGA-56Mv gene products in body fluids, cells or in tissue biopsy. Inspecific embodiments, measurement of serum or cellular SGA-56M andvariants thereof, including SGA-56Mv protein levels can be made todetect or stage breast cancer, e.g., infiltrative ductal carcinoma.

The present invention also relates to methods for the identification ofsubjects having a predisposition to cancer, e.g., breast cancer. Thesubject can be any animal, but preferably the subject is a mammal, andmost preferably the subject is a human. In a non-limiting examplenucleic acid molecules of the invention can be used as diagnostichybridization probes or as primers for quantitative RT-PCR analysis todetermine expression levels of the SGA-56M gene products and variantsthereof, including SGA-56Mv. In another example, nucleic acid moleculesof the invention can be used as diagnostic hybridization probes or asprimers for diagnostic PCR analysis for the identification of SGA-56Mand variants thereof, including SGA-56Mv naturally occurring ornon-naturally occurring gene mutations, allelic variations andregulatory defects in SGA-56M and SGA-56Mv genes.

Imaging methods, for imaging the localization and/or amounts of SGA-56Mand variants thereof, including SGA-56Mv gene products in a patient, arealso provided for diagnostic and prognostic use.

Further, methods are presented for the treatment of cancer, includingbreast cancer. Such methods comprise the administration of compositionsthat are capable of modulating the level of SGA-56M and variantsthereof, including SGA-56Mv gene expression and/or the level of SGA-56Mand SGA-56Mv gene product activity in a subject. The subject can be anyanimal, preferably a mammal, more preferably a human.

Still further, the present invention relates to methods for the use ofthe SGA-56M gene and variants thereof, including SGA-56Mv, and/orSGA-56M and variants thereof, including SGA-56Mv gene products for theidentification of compounds that modulate SGA-56M or SGA-56Mv geneexpression and/or the activity of SGA-56M or SGA-56Mv gene products.Such compounds can be used as agents to prevent and/or treat breastcancer or any cancer wherein SGA-56M and variants thereof, includingSGA-56Mv, are expressed at levels that are higher than those detected incorresponding normal tissue. Such compounds can also be used to palliatethe symptoms of the disease, and control the metastatic potential ofbreast cancer or any cancer wherein SGA-56M and variants thereof,including SGA-56Mv, are expressed at levels higher than those observedin corresponding normal tissue.

The invention also provides methods of preventing cancer byadministering the product of the SGA-56M gene and variants thereof,including SGA-56Mv or a fragment of the SGA-56M gene product andvariants thereof, including SGA-56Mv in an amount effective to elicit animmune response in a subject. The subject can be any animal, preferablya mammal, more preferably a human. The invention also provides methodsof treating or preventing cancer by administering the nucleic acid thatencodes the SGA-56M gene product and variants thereof, includingSGA-56Mv or a fragment of the nucleic acid that encodes the SGA-56M orSGA-56Mv gene product in an amount effective to elicit an immuneresponse. The invention further provides methods of treating orpreventing cancer by administering a protein or a peptide encoded by theSGA-56M gene and variants thereof, including SGA-56Mv in an amounteffective to elicit an immune response. The immune response can beeither humoral or cellular or both. In a preferred embodiment theinvention provides a method of immunizing against breast or lung cancer.

The invention relates to screening assays to identify antagonists oragonists of the SGA-56M gene or gene product and variants thereof,including SGA-56Mv. Thus, the invention relates to methods ofidentifying agonists or antagonists of the SGA-56M gene or gene productand variants thereof, including SGA-56Mv and the use of said agonist orantagonist to treat or prevent breast cancer or other types of cancer.

The invention also provides methods of treating cancer by providingtherapeutic amounts of an anti-sense nucleic acid molecule. Ananti-sense nucleic molecule is a nucleic acid molecule that is thecomplement of all or a part of the SGA-56M or SGA-56Mv gene sequences orSGA-56M and SGA-56Mv ORFs and which therefore can hybridize to theSGA-56M gene and variants thereof, including SGA-56Mv or a fragmentthereof. Hybridization of the anti-sense molecule can inhibit expressionof the SGA-56M or SGA-56Mv gene. In a preferred embodiment the method isused to treat breast cancer.

The invention also includes a kit for assessing whether a patient isafflicted with breast cancer or other types of cancer. This kitcomprises reagents for assessing expression of an SGA-56M or SGA-56Mvgene product.

In another aspect, the invention relates to a kit for assessing thesuitability of each of a plurality of compounds for inhibiting cancerincluding breast cancer in a patient. The kit comprises a reagent forassessing expression of an SGA-56M or SGA-56Mv gene products, and mayalso comprise a plurality of compounds.

In another aspect, the invention relates to a kit for assessing thepresence of cancer cells. This kit comprises an antibody, wherein theantibody binds specifically with a protein corresponding to an SGA-56Mgene product and variants thereof, including SGA-56Mv. The kit may alsocomprise a plurality of antibodies, wherein the plurality bindsspecifically with different epitopes on an SGA-56M gene product andvariants thereof, including SGA-56Mv.

The invention also includes a kit for assessing the presence of cancercells, wherein the kit comprises a nucleic acid (e.g., oligonucleotide)probe. The probe binds specifically with a transcribed polynucleotidecorresponding to an SGA-56M gene product and variants thereof, includingSGA-56Mv. The kit may also comprise a plurality of probes, wherein eachof the probes binds specifically with a transcribed polynucleotidecorresponding to a different mRNA sequence transcribed from the SGA-56Mgene and variants thereof, including SGA-56Mv.

Kits for diagnostic use, comprising in a container, primers for use inPCR that can amplify SGA-56M cDNA and variants thereof, including theSGA-56Mv cDNA and/or genes and, in a separate container, a standardamount of SGA-56M or SGA-56Mv cDNA are also provided.

The invention also provides transgenic non-human animals (e.g., mice)that express SGA-56M or SGA-56Mv nucleic acids and proteins encoded by atransgene. Transgenic, non-human knockout animals (e.g., mice), in whichan SGA-56M gene and variants thereof, including SGA-56Mv has beenpartially or completely inactivated, are also provided.

Accordingly, the present invention provides a method for diagnosing acancer in a subject comprising detecting or measuring an SGA-56M orSGA-56Mv gene product in a sample derived from said subject, whereinsaid SGA-56M or SGA-56Mv gene product is (a) an RNA corresponding to SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; (b) a proteincomprising SEQ ID NO: 5 or SEQ ID NO: 6; (c) a nucleic acid comprising asequence hybridizable to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 4, or a complement thereof, under conditions of high stringency,or a protein comprising a sequence encoded by said hybridizablesequence; (d) a nucleic acid at least 90% homologous to SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complement thereof, asdetermined using the NBLAST algorithm, or a protein encoded thereby; inwhich elevated expression levels of an SGA-56M gene product and/orvariants thereof, including SGA-56Mv, relative to those of anon-cancerous sample or a pre-determined standard value for anoncancerous sample, indicate the presence of cancer in the subject. Inone embodiment of the foregoing diagnostic method, the subject is ahuman. In another embodiment, the cancer is breast or lung cancer. Inyet other embodiments, the sample is a tissue sample, a plurality ofcells, or a bodily fluid.

The present invention further provides methods for staging a cancer in asubject comprising detecting or measuring an SGA-56M gene product and/orvariants thereof, including SGA-56Mv, in a sample derived from saidsubject, wherein said SGA-56M gene product and/or variants thereof,(e.g., SGA-56Mv) is (a) an RNA corresponding to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 4; (b) a protein comprising SEQ ID NO: 5or SEQ ID NO: 6; (c) a nucleic acid comprising a sequence hybridizableto SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or acomplement thereof, under conditions of high stringency, or a proteincomprising a sequence encoded by said hybridizable sequence; (d) anucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complement thereof, underconditions of high stringency, or a protein comprising a sequenceencoded by said hybridizable sequence; (e) a nucleic acid at least 90%homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4,or a complement thereof, as determined using the NBLAST algorithm, or aprotein encoded thereby; in which elevated expression levels of theSGA-56M gene product and/or variants thereof, including SGA-56Mv,relative to those of a non-cancerous sample or a pre-determined standardvalue for a noncancerous sample, indicate an advanced stage of cancer inthe subject.

The present invention further provides methods for the treatment of acancer in a subject, comprising administering to the subject atherapeutically effective amount of a compound for treating the cancerthat antagonizes an SGA-56M gene product and/or variants thereof,including SGA-56Mv, wherein said SGA-56M or SGA-56Mv gene product is (a)an RNA corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 4; (b) a protein comprising SEQ ID NO: 5 or SEQ ID NO: 6; (c) anucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complement thereof, underconditions of high stringency, or a protein comprising a sequenceencoded by said hybridizable sequence; or a protein comprising asequence encoded by said hybridizable sequence; (d) a nucleic acid atleast 90% homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 4, or a complement thereof, as determined using the NBLASTalgorithm; or a protein encoded thereby. In one embodiment, the geneproduct whose expression is down-regulated is a protein encoded by anucleic acid comprising a nucleotide sequence with at least 90% sequenceidentity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. Inanother embodiment, the compound decreases expression of an RNAcorresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4. The antagonist can be (i) a protein; (ii) a peptide; (iii) an organicmolecule with a molecular weight of less than 500 daltons; (iv) aninorganic molecule with a molecular weight of less than 500 daltons; (v)an antisense oligonucleotide molecule that binds to said RNA andinhibits translation of said RNA; (vi) a ribozyme molecule that targetssaid RNA and inhibits translation of said RNA; (vii) an antibody thatspecifically/selectively binds to an SGA-56M gene product and variantsthereof, including SGA-56Mv; or (viii) a double stranded oligonucleotidethat forms a triple helix with a promoter of an SGA-56M gene andvariants thereof, including SGA-56Mv, wherein said SGA-56M and SGA-56Mvgene is a nucleic acid at least 90% homologous to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complement thereof, asdetermined using the NBLAST algorithm. In an embodiment wherein thecompound is an antibody, the antibody immunospecifically binds to aprotein comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:6.

The present invention further provides methods for vaccinating a subjectagainst cancer comprising administering to the subject a molecule thatelicits an immune response to an SGA-56M and/or SGA-56Mv gene product,wherein said SGA-56M and/or SGA-56Mv gene product is (a) an RNAcorresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4; (b) a protein comprising SEQ ID NO: 5 or SEQ ID NO: 6 (c) a nucleicacid comprising a sequence hybridizable to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3 or SEQ ID NO: 4, or a complement thereof, under conditionsof high stringency, or a protein comprising a sequence encoded by saidhybridizable sequence; (d) a nucleic acid at least 90% homologous to SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complementthereof, as determined using the NBLAST algorithm, or a protein encodedthereby. In one embodiment, the immune response is a cellular immuneresponse. In another embodiment, the immune response is a humoral immuneresponse. In yet another embodiment, the immune response is both acellular and a humoral immune response. Such immune responses conferprotective immunity against a cancer to a patient. Protective immunityrefers to a reduced risk for developing a cancer and, therefore,encompasses a partial or complete immunity.

The present invention yet further provides methods for determining riskof developing cancer in a subject, said method comprising (I) measuringan amount of an SGA-56M and/or SGA-56Mv gene product in a sample derivedfrom the subject, wherein said SGA-56M and/or SGA-56Mv gene product is:(a) an RNA corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 4; (b) a protein comprising SEQ ID NO: 5 or SEQ ID NO: 6; (c)a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complement thereof, underconditions of high stringency, or a protein comprising a sequenceencoded by said hybridizable sequence; (d) a nucleic acid at least 90%homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4,or a complement thereof, as determined using the NBLAST algorithm, or aprotein encoded thereby; and (II) comparing the amount of said SGA-56Mand/or SGA-56Mv gene product in the subject with the amount of SGA-56Mand/or SGA-56Mv gene product present in a non-cancerous sample orpredetermined standard for a noncancerous sample, wherein an elevatedamount of said SGA-56M or SGA-56Mv gene product in the subject relativeto the amount in the non-cancerous sample or pre-determined standard fora noncancerous sample indicates a risk of developing cancer in thesubject.

The present invention yet further provides methods for determining if asubject suffering from cancer is at risk for metastasis of said cancer,said method comprising measuring an amount of an SGA-56M and/or SGA-56Mvgene product in a sample derived from the subject, wherein said geneproduct is (a) an RNA corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 4; (b) a protein comprising SEQ ID NO: 5 or SEQID NO: 6; (c) a nucleic acid comprising a sequence hybridizable to SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complementthereof, under conditions of high stringency, or a protein comprising asequence encoded by said hybridizable sequence; (d) a nucleic acid atleast 90% homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 4, or a complement thereof, as determined using the NBLASTalgorithm, or a protein encoded thereby, wherein an elevated amount ofSGA-56M or SGA-56Mv gene products in the subject compared to the amountin the non-cancerous sample, or in the sample from the subject with thenon-metastasizing cancer, or the amount in the predetermined standardfor a noncancerous or non-metastasizing sample, indicates an increasedrisk for developing metastasis of said cancer in the subject.

The present invention yet further provides methods for screening toidentify a compound capable of binding to an SGA-56M or SGA-56Mvmolecule, said method comprising (I) contacting the SGA-56M or SGA-56Mvmolecule with a candidate agent, wherein said SGA-56M or SGA-56Mvmolecule is (a) an RNA corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 4; (b) a protein comprising SEQ ID NO: 5 or SEQID NO: 6; (c) a nucleic acid comprising a sequence hybridizable to SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complementthereof, under conditions of high stringency, or a protein comprising asequence encoded by said hybridizable sequence; (d) a nucleic acid atleast 90% homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 4, or a complement thereof, as determined using the NBLASTalgorithm, or a protein encoded thereby and (II) determining if thecandidate agent binds the SGA-56M or SGA-56Mv molecule. The screeningassay can be performed in vitro. In one embodiment, the SGA-56Mmolecule, and/or variants thereof (e.g., SGA-56Mv) is anchored to asolid phase. In another embodiment, the candidate agent is anchored to asolid phase. In other embodiments, the screening assay is performed insolution. In yet other embodiments, the SGA-56M molecule or variantthereof (e.g., SGA-56Mv) is expressed on the surface of a cell or in thecytosol of a cell in step (I). In other embodiments, the SGA-56Mmolecule or variant thereof (e.g., SGA-56Mv) is expressed endogenouslyin the cell; alternatively, the cell can be engineered to expressexogenous SGA-56M and/or variants thereof. In the foregoing screeningmethods, the candidate agent is preferably labeled, for exampleradioactively or enzymatically.

The present invention also encompasses a method for screening toidentify a protein capable of interacting with an SGA-56M gene productcomprising contacting an SGA-56M gene product to a plurality ofpolypeptides, wherein the SGA-56M gene product is: an RNA correspondingto SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; a proteincomprising SEQ ID NO:5 or SEQ ID NO:6; a nucleic acid comprising asequence hybridizable to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQID NO:4, or a complement thereof, under conditions of high stringency,or a protein comprising a sequence encoded by said hybridizablesequence; a nucleic acid at least 90% homologous to SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO:3, or SEQ ID NO:4, or a complement thereof, asdetermined using an NBLAST algorithm, or a protein encoded thereby; or anucleic acid sequence encoding a protein comprising SEQ ID NO:5 or SEQID NO:6; and determining if at least one protein binds to or forms acomplex with the SGA-56M gene product. Such methods may be performed invitro or in vivo, for example, in a cell. In one embodiment, the methodfor screening is a two-hybrid screening method which is generallyperformed in yeast cells.

The present invention provides methods for screening to identify aprotein or peptide that interacts with an SGA-56M or SGA-56Mv geneproduct, said method comprising (I) immunoprecipitating the SGA-56M orSGA-56Mv gene product from a cell lysate, wherein said SGA-56M orSGA-56Mv gene product is (a) an RNA corresponding to SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; (b) a protein comprising SEQ IDNO: 5 or SEQ ID NO: 6; (c) a nucleic acid comprising a sequencehybridizable to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4, or a complement thereof, under conditions of high stringency, or aprotein comprising a sequence encoded by said hybridizable sequence; (d)a nucleic acid at least 90% homologous to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3 or SEQ ID NO: 4, or a complement thereof, as determinedusing the NBLAST algorithm, or a protein encoded thereby; and (II)determining if at least one cellular protein binds to or forms a complexwith the SGA-56M or SGA-56Mv gene product in the immunoprecipitate.

The present invention yet further provides methods for screening toidentify a candidate agent capable of modulating (e.g., decreasing orincreasing) the expression level and/or activity of an SGA-56M gene,and/or variant thereof, such as SGA-56Mv, said method comprising (I)contacting said SGA-56M or SGA-56Mv gene with a candidate agent, whereinsaid SGA-56M or SGA-56Mv gene is a nucleic acid at least 90% homologousto SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 asdetermined using the NBLAST algorithm; and (II) measuring the level ofexpression and/or activity of an SGA-56M or SGA-56Mv gene product, saidSGA-56M or SGA-56Mv gene product selected from the group consisting ofan mRNA corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 4 or a protein comprising SEQ ID NO: 5 or SEQ ID NO: 6, whereinan increase or decrease in said level of expression and/or activityrelative to said level of expression and/or activity in the absence ofsaid candidate agent indicates that the candidate agent modulatesexpression of an SGA-56M or SGA-56Mv gene.

The present invention yet further provides an immunogenic compositioncomprising (I) a purified SGA-56M or SGA-56Mv gene product in an amounteffective at eliciting an immune response, wherein said gene product is(a) an RNA corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO: 4; (b) a protein comprising SEQ ID NO: 5 or SEQ ID NO: 6; (c)a nucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a complement thereof, underconditions of high stringency, or a protein comprising a sequenceencoded by said hybridizable sequence; (d) a nucleic acid at least 90%homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4,or a complement thereof, as determined using the NBLAST algorithm, or aprotein encoded thereby; and (II) a pharmaceutically acceptable carrieror excipient.

The present invention yet further provides a pharmaceutical compositioncomprising an antibody that specifically binds to a protein comprisingSEQ ID NO:5 or SEQ ID NO:6; and a pharmaceutically acceptable carrier.

The present invention yet further provides pharmaceutical compositionscomprising (I) an SGA-56M or SGA-56Mv gene product (e.g., SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4) or an antibody immunospecificfor a protein comprising SEQ ID NO:5 or SEQ ID NO:6; and (II) apharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention can beformulated, inter alia, for delivery as an aerosol, for parenteraldelivery, or for oral delivery.

The present invention yet further provides methods for diagnosing cancerin a subject comprising (I) administering to said subject a compoundthat binds specifically to a protein comprising the amino acid sequenceof SEQ D) NO: 5 or SEQ ID NO: 6, wherein said compound is bound to animaging agent; and (II) obtaining an internal image of said subject byvisualizing the compound bound to the imaging agent, wherein thedetection of the compound bound to the imaging agent provides a positiveindicator for diagnosing a cancer in the subject. In a preferredembodiment, the compound is an antibody. In a preferred mode of theembodiment, the antibody is conjugated to a radioactive metal and saidobtaining step comprises recording a scintographic image obtained fromthe decay of the radioactive metal.

Also provided are kits that are useful for practicing the methods of thepresent invention. In one embodiment, such a kit comprises, in one ormore containers, an oligonucleotide primer pair, wherein each primer iscomplementary to a different strand of a double-stranded nucleic acidsequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ IDNO: 4, wherein said primer pair is capable of priming a DNAamplification reaction; and, in a separate container, a reference DNAcomprising a purified double-stranded nucleic acid comprising SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In specific modes of theembodiment, each primer comprises a nucleotide sequence with at least 8,more preferably at least 10, yet more preferably at least 12, and mostpreferably at least 15 complementary nucleotides to complementarystrands of a double-stranded nucleic acid comprising SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

The present invention yet further provides a transgenic non-human animalwhich expresses from a transgene an SGA-56M or SGA-56Mv gene product,for example, an RNA corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3 or SEQ ID NO: 4, or a protein comprising SEQ ID NO: 5 or SEQ IDNO: 6.

The present invention yet further provides a method for testing theeffects of a candidate therapeutic compound comprising administeringsaid compound to a transgenic non-human animal which expresses anexogenous SGA-56M or SGA-56Mv gene product; and determining any effectsof said compound upon said transgenic non-human animal.

The present invention further provides host cells comprising nucleicacids encoding the polypeptides of the invention operably linked to apromoter, and methods of expressing such polypeptides by culturing thehost cells under conditions in which the nucleic acid molecule isexpressed.

3.1 Definitions

Specific or Selective: a nucleic acid used in a reaction, such as aprobe used in a hybridization reaction, a primer used in a PCR, or anucleic acid present in a pharmaceutical preparation, is referred to as“selective” if it hybridizes or reacts with the intended target morefrequently, more rapidly, or with greater duration than it does withalternative substances. Similarly, a polypeptide is referred to as“selective” if it binds an intended target, such as a ligand, hapten,substrate, antibody, or other polypeptide more frequently, more rapidly,or with greater duration than it does to alternative substances. Anantibody is referred to as “selective” if it binds via at least oneantigen recognition site to the intended target more frequently, morerapidly, or with greater duration than it does to alternativesubstances. A marker is selective to a particular cell or tissue type ifit is expressed predominantly in or on that cell or tissue type,particularly with respect to a biological sample of interest

Variant (s): A variant (v) of a polynucleotide or polypeptide, as theterm is used herein, is a polynucleotide or polypeptide that isdifferent from a reference polynucleotide or polypeptide, respectively.

Variant polynucleotides are generally limited so that the nucleotidesequence of the reference and the variant are closely related overalland, in many regions, identical. Changes in the nucleotide sequence ofthe variant may be silent. That is, they may not alter the amino acidsequence encoded by the polynucleotide. Where alterations are limited tosilent changes of this type a variant will encode a polypeptide with thesame amino acid sequence as the reference. Alternatively, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions, and truncations in the polypeptide encoded by the referencesequence.

Variant polypeptides are generally limited so that the sequences of thereference and the variant are similar overall and, in many regions,identical. For example, a variant and reference polypeptide may differin amino acid sequence by one or more substitutions (conservative ornon-conservative), additions, deletions, fusions, and truncations, whichmay be present or absent in any combination.

Correspond or Corresponding: Between nucleic acids, “corresponding”means homologous to or complementary to a particular sequence or portionof the sequence of a nucleic acid. As between nucleic acids andpolypeptides, “corresponding” refers to amino acids of a peptide encodedby the nucleic acid sequence or a portion thereof or a complement ofeither. As between polypeptides (e.g., peptides, polypeptides, orproteins), “corresponding” refers to an amino acid sequence of a firstpolypeptide that is identical or homologous to an amino acid sequence ofa second polypeptide.

SGA-56M GENE PRODUCT: As used herein, unless otherwise indicated, anSGA-56M gene product is: an RNA corresponding to SEQ ID NO: 1 or SEQ IDNO: 2; a protein comprising SEQ ID NO: 5; a nucleic acid sequenceencoding an amino acid sequence comprising SEQ ID NO: 5; a nucleic acidcomprising a sequence hybridizable to SEQ ID NO: 1 or SEQ ID NO: 2, orcomplement thereof, under conditions of high stringency, or a proteincomprising a sequence encoded by said hybridizable sequence; a nucleicacid at least 90% homologous to SEQ ID NO: 1 or SEQ ID NO: 2, or acomplement thereof, as determined using the NBLAST algorithm; a nucleicacid at least 90% homologous to SEQ ID NO: 1 or SEQ ID NO: 2, or acomplement thereof, or a fragment or derivative of any of the foregoingproteins or nucleic acids.

SGA-56Mv GENE PRODUCT: As used herein, unless otherwise indicated, anSGA-56Mv gene product is: an RNA corresponding to SEQ ID NO: 3 or SEQ IDNO: 4; a protein comprising SEQ ID NO: 6; a nucleic acid sequenceencoding an amino acid sequence comprising SEQ ID NO: 6; a nucleic acidcomprising a sequence hybridizable to SEQ ID NO: 3 or SEQ ID NO: 4, or acomplement thereof, under conditions of high stringency, or a proteincomprising a sequence encoded by said hybridizable sequence; a nucleicacid at least 90% homologous to SEQ ID NO: 3 or SEQ ID NO: 4, or acomplement thereof, as determined using the NBLAST algorithm; a nucleicacid at least 90% homologous to SEQ ID NO: 3 or SEQ ID NO: 4 or afragment or derivative of any of the foregoing proteins or nucleicacids.

CONTROL ELEMENTS: As used herein refers collectively to promoterregions, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control elements needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell.

PROMOTER REGION: Is used herein in its ordinary sense to refer to anucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

OPERABLY LINKED: As used herein refers to an arrangement of elementswherein the components so described are configured so as to performtheir usual function. Thus, control elements operably linked to a codingsequence are capable of effecting the expression of the coding sequence.The control elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof.

MODULATE: As used herein, a compound which is capable of increasing ordecreasing the level and/or activity of an SGA-56M and/or SGA-56Mvmolecule may be referred to herein as an SGA-56M and/or SGA-56Mvmodulator.

ANTAGONIST: As used herein, a compound capable of reducing the leveland/or activity of an SGA-56M and/or SGA-56Mv molecule or a variantthereof may be referred to herein as an SGA-56M and/or SGA-56Mvantagonist.

AGONIST: As used herein, a compound capable of increasing the leveland/or activity of an SGA-56M and/or SGA-56Mv molecule or a variantthereof may be referred to herein as an SGA-56M and/or SGA-56Mv agonist.

SGA-56M AND/OR SGA-56Mv ACTIVITY: As used herein, the term “SGA-56Mand/or SGA-56Mv activity” refers to an activity of SGA-56M and/orSGA-56Mv which contributes to the onset, progression, and/or metastaticspread of breast or lung cancer and/or other cancers.

ELEVATED SGA-56M and/or SGA-56Mv LEVELS: As used herein the terms“elevated”, “over-expressed”, “up-regulated”, or “increased” SGA-56Mand/or SGA-56Mv levels refer to an approximately two-fold or greaterincrease in the expression of SGA-56M and/or SGA-56Mv transcript and/orprotein as compared to or relative to that of a control tissue, whichexpresses a baseline level of SGA-56M and/or SGA-56Mv. As used herein,the terms control tissue, non-cancerous sample, or predeterminedstandard for a noncancerous sample may be used interchangeably to referto a tissue that expresses a baseline level of SGA-56M and/or SGA-56Mv.

IMMUNOGICALLY SPECIFIC FOR: Antibodies which are immunologicallyspecific for SGA-56M and/or SGA-56Mv are capable of recognizing SGA-56Mand/or SGA-56Mv largely to the exclusion of other molecules.

SPECIFIC BINDING PAIR: A member of a specific binding pair (“sbpmember”) refers to one of two different molecules, having an area on thesurface or in a cavity which specifically binds to and is therebydefined as complementary with a particular spatial and polarorganization of another molecule. The members of the specific bindingpair are referred to as ligand and receptor (antiligand). These may bemembers of an immunological pair such as antigen-antibody, or may beoperator-repressor, nuclease-nucleotide, biotin-avidin, hormone-hormonereceptor, IgG-protein A, DNA-DNA, DNA-RNA, and the like.

CONSISTING ESSENTIALLY OF: The phrase “consisting essentially of” whenreferring to a particular nucleotide or amino acid means a sequencehaving the properties of a given SEQ ID NO:. For example, when used inreference to an amino acid sequence, the phrase includes the sequenceper se and molecular modifications that would not affect the basic andnovel characteristics of the sequence.

PRIMER PAIR: As used herein, the terms “primer pair” or “oligonucleotideprimer pair” when used in the context of a polymerase chain reaction(PCR), for example, refer to a first and a second primer of sufficientcomplementarity to a template nucleic acid sequence to hybridize to thetemplate nucleic acid sequence at two physically separated sites and onseparate strands such that extension from a first primer produces asingle stranded nucleic acid which is at least partially complementaryto a single stranded nucleic acid extended from a second primer.

PROTECTIVE IMMUNITY: As used herein, the terms “protective immunity” or“protective immune response” are intended to mean that the vaccinatedsubject mounts an active immune response to the antigen administered(i.e., an SGA-56M or SGA-56Mv molecule), such that upon subsequentexposure to the antigen or a cell expressing the antigen (e.g., a cancercell), the subject is able to mount an immune response specific for theantigen or cell expressing the antigen. Such an immune response reducesthe number of antigen positive cells in a subject. Thus, a protectiveimmune response will decrease the incidence of cancer in a vaccinatedsubject.

FUNCTIONAL FRAGMENT: As used herein, a functional fragment of a nucleicor amino acid molecule of the invention is a fragment which retains somefunctional property of the larger nucleic or amino acid molecule.Examples of such functional properties include: coding for a functionalpolypeptide (for a nucleic acid fragment), binding to proteins, or theability to mediate changes in cellular behavior associated with SGA-56Mand/or SGA-56Mv expression, such as, for example, changes in cellmorphology, cell division, differentiation, adhesion, motility,phosphorylation, or dephosphorylation of cellular proteins. One ofordinary skill in the art can readily determine using the assaysdescribed herein and those well known in the art to determine whether afragment is a functional fragment of a nucleic or amino acid moleculeusing no more than routine experimentation.

The basic molecular biology techniques used to practice the methods ofthe invention are well known in the art, and are described for examplein Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York; Ausubel et al., 1988, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York; and Ausubelet al., 2002, Short Protocols in Molecular Biology, John Wiley & Sons,New York).

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B show the nucleic acid sequence of the 2917 bp SGA-56Mtranscript comprising a coding sequence (CDS) spanning 225-2630 bp andthe amino acid sequence encoded therefrom.

FIGS. 2A and B shows the nucleic acid sequence of the 2779 bp SGA-56Mvtranscript comprising a CDS spanning 225-2492 bp and the amino acidsequence encoded therefrom. SGA-56Mv contains an in-frame deletionspanning 925-1063 bp of SGA-56M, as illustrated in FIG. 1.

FIG. 3 shows a photograph of an agarose gel of RT-PCR productsvisualized by ethidium bromide staining and generated bysemi-quantitative RT-PCR of normal vs. transformed breast cells. Aregion common to both SGA-56M and SGA-56Mv cDNA was amplified in thisassay. Samples are loaded as follows: (1) MCF-7, (2) T47D, (3) normalhuman mammary epithelial cells, (4) SKBR-3, (5) Hs578T, (6) MDA-MB231,(7) MDA-MB435s, (8) MDA-MB453, (9) H3396, and (10) BT549. The controlgene EF-1 was included for comparison.

FIG. 4 shows a photograph of an agarose gel of RT-PCR productsvisualized by ethidium bromide staining and generated bysemi-quantitative RT-PCR of various tumor cell-lines. A region common toboth SGA-56M and SGA-56Mv cDNA was amplified in this assay. Samples areloaded as follows: (1) HCT-15, (2) HCT-116, (3) HT-29, (4) RCA, (5)NCI-H23, (6) NCI-H460, (7) NCI-H226, (8) MiaPaCa-2, (9) Bx-PC3, (10)CAPAN-2, (11) WM-115, (12) SK-MEL5, (13) SK-MEL28, (14) Colo-853, (15)Colo-857, and (16) GRM. The control gene EF-1 was included forcomparison.

FIGS. 5A and B show hybridization patterns of SGA-56M/SGA-56Mv and EF-1normal tissue expression levels on a Multiple Tissue Expression (MTE™)Array. A region common to both SGA-56M and SGA-56Mv cDNA was amplifiedand used as a probe for this experiment (B). The control gene EF-1 wasincluded for comparison (A).

FIG. 6 shows a hybridization pattern of SGA-56M and SGA-56Mv on theCancer Profiling Array (CPA™). A cancer-selective expression pattern isrevealed. A region common to both SGA-56M and SGA-56Mv cDNA wasamplified and used as a probe for this experiment.

FIG. 7 shows an amino acid sequence of the SGA-56M protein encoded bySEQ ID NO: 1, which comprises the CDS (SEQ ID NO: 2). A putativetransmembrane region (TM) is indicated in bold and underlined spanningamino acids 340-361.

FIG. 8 shows an amino acid sequence of the SGA-56Mv protein encoded bySEQ ID NO: 3, which comprises the CDS (SEQ ID NO: 4). A putativetransmembrane region (TM) is indicated in bold and underlined spanningamino acids 294-315.

FIG. 9 shows a comparison of SGA-56M and SGA-56Mv proteins with putativetransmembrane regions (TMs) illustrated. SGA-56Mv includes an in-framedeletion of amino acids 234-279 of SGA-56M.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that the SGA-56M andSGA-56Mv genes are over-expressed in cancer cells and tissues such asbreast cancer cells. The invention relates to methods of using theSGA-56M gene and variants thereof, including SGA-56Mv, and/or theSGA-56M or SGA-56Mv gene products to diagnose, treat and prevent cancer,e.g., breast cancer. The invention further relates to methods of usingthe SGA-56M or SGA-56Mv genes or SGA-56M or SGA-56Mv gene products toevaluate the prognosis of a patient diagnosed with cancer. The inventionalso relates to the discovery that the SGA-56M or SGA-56Mv genes areover-expressed in metastatic cancer cells. Thus, the inventioncontemplates the use of the SGA-56M gene and variants thereof, includingSGA-56Mv, and/or gene products to evaluate a cancer patient's risk ofmetastasis of a cancer, e.g., breast cancer.

In the development of breast neoplasia and other cancers, there is asubset of genes that are specifically expressed at various stages, and acertain number of these will be critical for the progression ofmalignancy, especially those associated with the metastatic spread ofthe disease. As described by way of example, infra, genes whoseexpression is associated with breast carcinomas at various stages ofneoplastic development, were identified using Suppression SubtractiveHybridization (SSH) and high-throughput cDNA microarray analysis (Chu etal., 1997, Proc. Natl. Acad. Sci. U.S.A. 94(19): 10057; Kuang et al.,1998, Nuc. Acids Res. 26(4): 1116). As described herein, SSH generatedcDNA libraries derived from the breast cancer cell line MCF-7 werescreened using microarrays for genes which were expressed at elevatedlevels in the cancerous MCF-7 cells as compared to normal human mammaryepithelial cells (HMECs). A total of 1536 clones were screened. Thenovel SGA-56M gene identified herein, and several previously identifiedbreast cancer associated genes were identified using this approach.Details concerning the isolation and characterization of the SGA-56McDNA and variants thereof, including SGA-56Mv, and their expressionpatterns in cancer cell lines and tissues is described in detail in theexamples provided infra

The present invention encompasses methods for the diagnosis, prognosisand staging of breast cancer and other cancers, e.g., by the monitoringof the effect of a therapeutic treatment. Further provided are methodsfor the use of the SGA-56M or SGA-56Mv genes and/or SGA-56M or SGA-56Mvgene products in the identification of compounds that modulate theexpression of the SGA-56M or SGA-56Mv gene or the activity of theSGA-56M or SGA-56Mv gene product. Expression of the SGA-56M gene andvariants thereof including SGA-56Mv, is upregulated in various types ofcancer cells including breast cancer cell lines and tissues. As such,the SGA-56M or SGA-56Mv gene products can be involved in the mechanismsunderlying the onset and development of breast cancer and other types ofcancer as well as the regional infiltration and metastatic spread ofcancer. Thus, the present invention also provides methods for theprevention and/or treatment of breast cancer and other types of cancer,and for the control of metastatic spread of breast cancer and othertypes of cancer. Such methods are based on modulation of the expressionand/or activity of the SGA-56M and/or SGA-56Mv gene or gene product.

The invention further provides for screening assays and methods ofidentifying agonists and antagonists of the SGA-56M or SGA-56Mv gene orgene product. The invention also provides methods of vaccinating anindividual against cancer, including breast cancer, by administering anamount of the SGA-56M or SGA-56Mv gene, gene product, or fragmentthereof, in an amount that effectively elicits an immune response in asubject who has cancer or is at risk of developing cancer, includingbreast cancer.

5.1. The SGA-56M and SGA-56Mv Genes

Nucleotide sequences that encode the SGA-56M and SGA-56Mv gene openreading frames are described herein. The SGA-56M DNA (2917 bp) SEQ IDNO: 1 was cloned by PCR using gene-specific primers. The SGA-56Mv DNA(2779 bp) SEQ ID NO: 3 was cloned by PCR using gene-specific primers.The SGA-56M DNA sequence comprises an open reading frame SEQ ID NO: 2spanning 225-2630 bp within SEQ ID NO: 1 that encodes a protein of 802amino acids (SEQ ID NO: 5). The SGA-56Mv DNA sequence comprises an openreading frame SEQ ID NO: 4 spanning 225-2492 bp within SEQ ID NO: 3 thatencodes a protein of 756 amino acids (SEQ ID NO: 6). SGA-56Mv is avariant form of SGA-56M that contains an in-frame deletion of 234-279amino acids from within SEQ ID NO: 5. As described in detail in section6, SGA-56M and SGA-56Mv share GenBank sequence homology with existingentries at both the nucleic acid or amino acid level using the NBLASTalgorithm (www.ncbi.nlm.nih.gov).

The SGA-56M or SGA-56Mv nucleic acids and derivatives used in thepresent invention include but are not limited to RNA corresponding toSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; a nucleic acidcomprising a sequence hybridizable to SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3 or SEQ ID NO: 4, or the complement of any of the foregoing nucleicacids; a nucleic acid at least 90% homologous to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or at least 90% homologous thecomplement of any of the foregoing nucleic acids (e.g., as determinedusing the NBLAST algorithm under default parameters). As used herein an“RNA corresponding to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ IDNO: 4 means an RNA comprising a sequence that is the same or the(inverse) complement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQID NO: 4, except that thymidines (T's) can be replaced with uridines(U's). A nucleic acid derived from such RNA includes but is not limitedto cDNA of said RNA, and cRNA (e.g., RNA that is derived from said cDNA;see, e.g., U.S. Pat. Nos. 5,545,522; 5,891,636; 5,716,785). In thepresent invention, hybridizability can be determined under low,moderate, or high stringency conditions and preferably is underconditions of high stringency.

The SGA-56M or SGA-56Mv protein and derivatives used in the presentinvention include, but are not limited to proteins (and other molecules)comprising SEQ ID NO: 5 or SEQ ID NO: 6, proteins comprising a sequenceencoded by a nucleic acid hybridizable to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3 or SEQ ID NO: 4 or their complements, and proteins encodedby a nucleic acid at least 90% homologous to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3 or SEQ ID NO: 4, or their complement, e.g., as determinedusing the NBLAST algorithm.

The SGA-56M or SGA-56Mv nucleic acids used in the present inventioninclude but are not limited to (a) a DNA comprising the DNA sequenceshown in FIG. 1 (SEQ ID NO: 1), FIG. 2 (SEQ ID NO: 3), or a complementthereof; (b) any DNA sequence that hybridizes to the DNA sequences or acomplement thereof that encode the amino acid sequences shown in FIG. 7and FIG. 8, under low, moderate or highly stringent conditions, asdisclosed infra in Section 5.1.1; as well as proteins encoded by suchnucleic acids. In a specific embodiment, nucleic acids used in theinvention encode a gene product that has at least one conservative orsilent substitution. The encoded proteins are also provided for use.Additional molecules that can be used in the invention include, but arenot limited to, protein derivatives that can be made by altering theirsequences by substitutions, additions or deletions, and their encodingnucleic acids. Due to the degeneracy of nucleotide coding sequences,other DNA sequences that encode substantially the same amino acidsequence as a component gene or cDNA can be used in the practice of thepresent invention. These include but are not limited to nucleotidesequences comprising all or portions of the component protein gene thatare altered by the substitution of different codons that encode afunctionally equivalent amino acid residue within the sequence, thusproducing a silent change. Likewise, the derivatives of the inventioninclude, but are not limited to, those containing, as a primary aminoacid sequence, part or all of the amino acid sequence of a componentprotein, including altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a conservative change. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity (a “conservative amino acid substitution”) that actsas a functional equivalent, resulting in a conservative alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

The invention includes the use of the SGA-56M or SGA-56Mv specificoligonucleotide sequences which preferably hybridize under highlystringent or moderately stringent conditions as described infra inSection 5.1.1 to at least about 6, preferably about 12, more preferablyabout 18, consecutive nucleotides of the SGA-56M or SGA-56Mv genesequences described above as being useful for the detection of anSGA-56M or SGA-56Mv gene product for the diagnosis and prognosis ofcancer. Such gene products include, e.g., an RNA corresponding to SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; a nucleic acidcomprising a sequence hybridizable to SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3 or SEQ ID NO: 4, or its complement under conditions of highstringency; a nucleic acid at least 90% homologous to SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 or its complement as determinedusing the NBLAST algorithm.

The invention also includes the use of nucleic acid molecules,preferably DNA molecules, that preferably hybridize under highlystringent or moderately stringent conditions as described infra inSection 5.1.1 to, and are therefore the inverse complements of, thenucleic acid sequences, described, inter alia, in Section 3 above. Thesenucleic acid molecules may encode or act as antisense molecules that maybe used to partially or completely inhibit SGA-56M and/or SGA-56Mvexpression. With respect to SGA-56M or SGA-56Mv gene regulation, suchtechniques can be used to modulate, for example, the phenotype andmetastatic potential of breast cancer or other cancer cells. Moreover,such sequences also provide components of ribozyme and/or triple helixsequences that may be used to advantage to modulate (e.g., inhibit)SGA-56M or SGA-56Mv gene expression. Thus, these sequences and ribozymeand/or triple helix sequences comprising such sequences may be used forthe treatment and/or prevention of cancer.

In one embodiment, the invention encompasses methods of using theSGA-56M or SGA-56Mv gene coding sequence or fragments and degeneratevariants of DNA sequences which encode the SGA-56M or SGA-56Mv gene orgene product, including naturally occurring and non-naturally occurringvariants thereof. A non-naturally occurring variant is one that isengineered by man. A naturally occurring SGA-56M or SGA-56Mv gene, geneproduct, or variant thereof is one that is not engineered by man. In themethods of the invention wherein an SGA-56M or SGA-56Mv gene product ina sample derived from a subject is detected or measured, naturallyoccurring SGA-56M or SGA-56Mv gene products are detected, including, butnot limited to wild-type SGA-56M or SGA-56Mv gene products as well asmutants, allelic variants, splice variants, polymorphic variants, etc.In general, such mutants and variants are believed to be highlyhomologous to SEQ ID NO: 1, or SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:4, e.g., at least 90% homologous and/or hybridizable under highstringency conditions. In specific embodiments, the mutants and variantsbeing detected or measured may comprise (or, if nucleic acids, encode)not more than 1, 2, 3, 4, or 5 point mutations (substitutions) relativeto SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 and/orcomprise or encode only conservative amino acid substitutions.

In other methods of the invention, wild-type, or naturally occurringvariant, or non-naturally occurring variant SGA-56M or SGA-56Mvsequences may be used in the methods of the invention (e.g., invaccination, immunization, antisense, or ribozyme procedures).

An SGA-56M or SGA-56Mv gene fragment may be a complementary DNA (cDNA)molecule or a genomic DNA molecule that may comprise one or moreintervening sequences or introns, as well as regulating regions locatedbeyond the 5′ and 3′ ends of the coding region or within an intron.

The present invention provides for methods of using isolated nucleicacid molecules encoding an SGA-56M or SGA-56Mv protein, polypeptide, orfragments, derivatives, and variants thereof that include, bothnaturally occurring and non-naturally occurring variants or mutants. Theinvention also contemplates, for use in the methods of the invention,the use of 1) any nucleic acid that encodes an SGA-56M or SGA-56Mvpolypeptide of the invention; 2) any nucleic acid that hybridizes to thecomplement of the sequences disclosed herein, preferably under highlystringent conditions as disclosed infra in Section 5.1.1, and encodes afunctionally equivalent gene product; and/or 3) any nucleic acidsequence that hybridizes to the complement of the sequences disclosedherein, preferably under moderately stringent conditions, as disclosedinfra in Section 5.1.1 yet which still encodes a gene product thatdisplays a functional activity of SGA-56M or SGA-56Mv.

As discussed above, the invention also contemplates the use of isolatednucleic acid molecules that encode a variant protein or polypeptide. Thevariant protein or polypeptide can occur naturally or non-naturally. Itcan be engineered by introducing nucleotide substitutions, e.g. pointmutations, or additions or deletions into the nucleotide sequence of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In a specificembodiment, one or more, but not more than 5, 10, or 25 amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. Following mutagenesis, the encodedprotein can be expressed recombinantly and the activity of the proteincan be determined.

In a specific embodiment, the invention provides for the use of SGA-56Mor SGA-56Mv derivatives and analogs of the invention which arefunctionally active, i.e., they are capable of displaying one or moreknown functional activities associated with a (wild-type) SGA-56M orSGA-56Mv-encoded protein. Such functional activities include but are notlimited to antigenicity/immunogenicity (ability to bind or compete withSGA-56M or SGA-56Mv for binding to an anti-SGA-56M or anti-SGA-56Mvantibody, respectively or ability to generate antibody which binds toSGA-56M or SGA-56Mv), ability to bind or compete with SGA-56M orSGA-56Mv for binding to other proteins or fragments thereof, such asproteins capable of forming complexes with SGA-56M and/or SGA-56Mv.

The nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3or SEQ ID NO: 4 or portions thereof, may be used, for example, ashybridization probes. Nucleic acid molecules encoding an SGA-56M orSGA-56Mv gene product can be isolated using standard hybridization andcloning techniques (See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 formethodological details pertaining to the methods of the invention.

In addition, gene products encoded by SGA-56M or SGA-56Mv, includingSGA-56M or SGA-56Mv peptide fragments can comprise components of fusionproteins, which may be used to facilitate recovery, detection, and/orlocalization of another protein of interest. Antibodies immunologicallyspecific for SGA-56M and/or SGA-56Mv may also be used effectively torecover, detect, and/or localize another protein of interest that, forexample, binds to SGA-56M and/or SGA-56Mv. In addition, genes and geneproducts encoded for by SGA-56M or SGA-56Mv can be used as researchreagents, e.g., for genetic mapping.

Additionally, the present invention contemplates use of the nucleic acidmolecules, polypeptides, and/or antagonists of gene products encoded forby the SGA-56M or SGA-56Mv gene to screen, diagnose, prevent and/ortreat disorders characterized by aberrant expression or activity of theSGA-56M or SGA-56Mv polypeptides, which include, cancers, such as, butnot limited to cancer of the breast, ovary, skin and lymphoid system.

The present invention encompasses the use of SGA-56M or SGA-56Mv nucleicacid molecules comprising cDNA, genomic DNA, introns, exons, promoterregions, 5′ and 3′ regulatory regions of the gene, RNA, hnRNA, mRNA,regulatory regions within RNAs, and degenerate variants thereof in themethods of the invention. Promoter sequences for SGA-56M or SGA-56Mv canbe determined by promoter-reporter gene assays and in vitro bindingassays.

In one embodiment, the invention comprises the use of a variant SGA-56Mor SGA-56Mv nucleic acid sequence that hybridizes to anaturally-occurring occurring variant SGA-56M or SGA-56Mv nucleic acidmolecule under stringent conditions as described infra in Section 5.1.1.In another embodiment, the invention contemplates the use of an SGA-56Mor SGA-56Mv variant nucleic acid sequence that hybridizes to anaturally-occurring occurring variant SGA-56M or SGA-56Mv nucleic acidmolecule under moderately stringent conditions as described infra inSection 5.1.1.

A nucleic acid molecule is intended to include DNA molecules (e.g.,cDNA, genomic DNA), RNA molecules (e.g., hnRNA, pre-mRNA, mRNA), and DNAor RNA analogs generated using nucleotide analogs. The nucleic acidmolecule can be single-stranded or double-stranded.

The SGA-56M or SGA-56Mv gene sequences used in the methods of theinvention are of human origin, however, homologs of SGA-56M or SGA-56Mvisolated from other mammals may also be used in the methods of theinvention. Thus, the invention also includes the use of SGA-56M orSGA-56Mv homologs isolated from non-human animals such as: non-humanprimates; rats; mice; farm animals including, but not limited to:cattle; horses; goats; sheep; pigs; etc.; household pets including, butnot limited to: cats; dogs; etc. in the methods of the invention.

Still further, such molecules may be used as components of diagnosticand/or prognostic methods whereby, for example, the presence of aparticular SGA-56M or SGA-56Mv allele or alternatively spliced SGA-56Mor SGA-56Mv transcript responsible for causing or predisposing one tobreast cancer or other cancers may be detected.

The invention also includes the use of transcriptional regulators thatcontrol the level of expression of an SGA-56M or SGA-56Mv gene product.A transcriptional regulator can include, e.g., a protein that binds aDNA sequence and up-regulates or down-regulates the transcription of theSGA-56M or SGA-56Mv gene. A transcriptional regulator can also include anucleic acid sequence that can be either upstream or downstream from theSGA-56M or SGA-56Mv gene and which binds an effector molecule thatenhances or suppresses SGA-56M or SGA-56Mv gene transcription.

Still further, the invention encompasses the use of SGA-56M or SGA-56Mvgene coding sequences or fragments thereof in screens to identifyproteins, peptides or nucleic acids related to the onset and/ormetastatic spread of cancer, including breast and lung cancer. Examplesof engineered yeast-based systems for investigating protein-proteininteractions include, but are not limited to, the yeast two-hybridsystem.

The invention also encompasses the use of (a) DNA vectors comprising anyof the foregoing SGA-56M or SGA-56Mv coding sequences and/or theircomplements (e.g., antisense); (b) DNA expression vectors comprising anyof the foregoing SGA-56M or SGA-56Mv coding sequences operatively linkedor associated with a regulatory element that directs the expression ofthe coding sequences; and (c) genetically engineered host cellscomprising any of the foregoing SGA-56M or SGA-56Mv coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. Cell lines and/orvectors which comprise and/or express SGA-56M or SGA-56Mv can be used toproduce an SGA-56M or SGA-56Mv gene product for use in the methods ofthe invention. Such methods include, e.g., vaccination against breastcancer or other cancers in which expression of SGA-56M or SGA-56Mv isfound to be elevated and screening assays to identify antagonists andagonists that bind and/or interact with SGA-56M and/or SGA-56Mv ormodulate (i.e., suppress or enhance) expression of SGA-56M and/orSGA-56Mv.

As used herein, regulatory elements include, but are not limited toinducible and non-inducible promoters, enhancers, tissue specificpromoters and/or enhancers, operators and other elements that drive andregulate expression and are known to those skilled in the art. Suchregulatory elements include but are not limited to the cytomegalovirus(hCMV) immediate early promoter, the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors.

The invention includes the use of fragments or derivatives of any of thenucleic acids disclosed herein in any of the methods of the invention.In various embodiments, a fragment or derivative comprises 10, 20, 50,100, 200, or more nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3.

In addition to the use of the SGA-56M or SGA-56Mv gene sequencesdescribed above, homologs of such sequences, exhibiting extensivehomology to the SGA-56M or SGA-56Mv gene product present in otherspecies can be identified and readily isolated, and used in the methodsof the invention without undue experimentation, by molecular biologicaltechniques well known in the art Further, there can exist homolog genesat other genetic loci within the genome that encode proteins that haveextensive homology to SGA-56M or SGA-56Mv. Such homologous genes, canencode multiple proteins, one or both of which are homologous to SGA-56Mand/or SGA-56Mv. Alternatively, such homologous genes can encode asingle protein with homology to SGA-56M and/or SGA-56Mv. Onceidentified, such genes can be used in the methods of the presentinvention. Still further, there can exist alternatively spliced variantsof the SGA-56M or SGA-56Mv gene. The invention thus includes the use ofany of homolog/ortholog/variant of SGA-56M and/or SGA-56Mv in themethods of the invention.

As an example, in order to clone a mammalian SGA-56M or SGA-56Mv genehomolog or variants using isolated human SGA-56M or SGA-56Mv genesequences as disclosed herein, such human SGA-56M or SGA-56Mv genesequences are labeled and used to screen a cDNA library constructed frommRNA obtained from appropriate cells or tissues (e.g., breast epithelialcells) derived from the organism of interest. With respect to thecloning of such a mammalian SGA-56M or SGA-56Mv homolog, a mammalianbreast cancer cell cDNA library may, for example, be used for screening.In one embodiment, such a screen would employ a probe corresponding toall or a portion of the SGA-56M or SGA-56Mv open reading frame SEQ IDNO: 2 or SEQ ID NO: 4. In yet another embodiment, such a screen wouldemploy one or more probes corresponding to all or a portion of thecoding sequence for SGA-56M (SEQ ID NO: 2) or SGA-56Mv (SEQ ID NO: 4),for example, a probe corresponding to the SGA-56M (SEQ ID NO: 1) orSGA-56Mv (SEQ ID NO: 3).

The hybridization and wash conditions used should be of a lowstringency, as described infra in Section 5.1.1 when the cDNA library isderived from a species other than the species from which the labeledsequence (e.g., probe) was derived.

Alternatively, the labeled fragment (or probe) may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions well known to those of skill in theart.

Further, an SGA-56M or SGA-56Mv gene homolog/ortholog/variant may beisolated from nucleic acid of an organism of interest by performing PCRusing two degenerate oligonucleotide primer pools based on amino acidsequences of SGA-56M and/or SGA-56Mv encoded gene products. The templatefor the reaction may, for example, be cDNA obtained by reversetranscription of mRNA prepared from, for example, mammalian cell linesor tissue known or suspected to express an allele, homolog, ortholog, orvariant of SGA-56M and/or SGA-56Mv.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an SGA-56M orSGA-56Mv-related nucleic acid sequence. The PCR fragment may then beused to isolate a larger fragment of an SGA-56M or SGA-56Mv-relatednucleic acid sequence (e.g., a full length cDNA clone) by a variety ofmethods. For example, the amplified fragment may be labeled and used toscreen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

PCR technology may be utilized to isolate additional nucleic acidsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (e.g., oneknown, or suspected, to express the SGA-56M or SGA-56Mv gene, such as,for example, breast cancer cell-lines). A reverse transcription reactionmay be performed on the RNA using an oligonucleotide primer specific orselective for the most 5′ end of the amplified fragment for the primingof first strand synthesis. The resulting RNA/DNA hybrid may then be“tailed” with guanines using a standard terminal transferase reaction,the hybrid may be digested with RNAase H, and second strand synthesismay then be primed with a poly-C primer. Thus, nucleic acid sequencesupstream of the amplified fragment may easily be isolated. For a reviewof PCR technology and cloning strategies which may be used, see, e.g.,PCR Primer, 1995, Dieffenbach et al., ed., Cold Spring Harbor LaboratoryPress; Sambrook et al., 1989, supra.

SGA-56M or SGA-56Mv gene coding sequences may additionally be used toisolate mutant SGA-56M or SGA-56Mv gene alleles. Such mutant alleles maybe isolated from individuals either known or susceptible to orpredisposed to have a genotype that contributes to the development ofcancer, e.g., breast cancer, including metastasis. Such mutant allelesmay also be isolated from individuals either known or susceptible to orpredisposed to have a genotype that contributes to resistance to thedevelopment of cancer, e.g., breast cancer, including metastasis. Mutantalleles and mutant allele products may then be utilized in thescreening, therapeutic and diagnostic methods and systems describedherein. Additionally, such SGA-56M or SGA-56Mv gene sequences can beused to detect SGA-56M or SGA-56Mv gene regulatory (e.g., promoter)defects that can affect the development and outcome of cancer. Mutantscan be isolated by any technique known in the art, e.g., PCR, screeninggenomic libraries, screening expression libraries.

As described below, the invention also relates to the use of an SGA-56Mor SGA-56Mv gene coding sequence or gene product in the methods of theinvention. An SGA-56M or SGA-56Mv gene coding sequence or gene productincludes, but is not limited to an RNA corresponding to SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, a protein comprising SEQ IDNO: 5 or SEQ ID NO: 6, or a nucleic acid comprising a sequencehybridizable to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4under conditions of high stringency, or a protein comprising a sequenceencoded by said hybridizable sequence or a nucleic acid at least 90%homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4as determined using the NBLAST algorithm or a protein encoded thereby.

5.1.1 Hybridization Conditions

A nucleic acid which is hybridizable to an SGA-56M or SGA-56Mv nucleicacid (e.g., having a sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 4 or to its reverse complement), or to anucleic acid encoding an SGA-56M or SGA-56Mv derivative, or to itsreverse complement under conditions of low stringency can be used in themethods of the invention to detect the presence of an SGA-56M orSGA-56Mv gene and/or presence or expression level of an SGA-56M orSGA-56Mv gene product. By way of example and not limitation, proceduresusing such conditions of low stringency are as follows (see also Shiloand Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792).Filters containing DNA are pretreated for 6 h at 40° C. in a solutioncontaining 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA,0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA.Hybridizations are carried out in the same solution with the followingmodifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe isused. Filters are incubated in hybridization mixture for 18-20 h at 40°C., and then washed for 1.5 h at 55° C. in a solution containing 2×SSC,25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency that may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

A nucleic acid which is hybridizable to an SGA-56M or SGA-56Mv nucleicacid (e.g., having a sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ED NO: 4 or to its reverse complement), or to anucleic acid encoding an SGA-56M or SGA-56Mv derivative, or to itsreverse complement under conditions of high stringency is also providedfor use in the methods of the invention. By way of example and notlimitation, procedures using such conditions of high stringency are asfollows. Prehybridization of filters containing DNA is carried out for 8h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. inprehybridization mixture containing 100 μg/ml denatured salmon sperm DNAand 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minbefore autoradiography. Other conditions of high stringency that may beused are well known in the art.

A nucleic acid which is hybridizable to an SGA-56M or SGA-56Mv nucleicacid (e.g., having a sequence as set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 4 or to its reverse complement), or to anucleic acid encoding an SGA-56M or SGA-56Mv derivative, or to itsreverse complement under conditions of moderate stringency is alsoprovided for use in the methods of the invention. For example, but notlimited to, procedures using such conditions of moderate stringency maybe performed according to the following method. Filters containing DNAare pretreated for 6 hours at 55° C. in a solution containing 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA.Hybridizations are carried out in the same solution with 5-20×10⁶ cpm³²P-labeled probe. Filters are incubated in hybridization mixture for18-20 hours at 55° C., and then washed twice for 30 minutes at 60° C. ina solution containing 1×SSC and 0.1% SDS. Filters are blotted dry andexposed for autoradiography. Washing of filters is done at 37° C. for 1hour in a solution containing 2×SSC, 0.1% SDS. Other conditions ofmoderate stringency that may be used are well known in the art. (see,e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; seealso, Ausubel et al., eds., in the Current Protocols in MolecularBiology series of laboratory technique manuals, 1987-1997 CurrentProtocols,© 1994-1997 John Wiley and Sons, Inc.).

5.2. Protein Products of the SGA-56M and SGA-56Mv Genes

In another embodiment, the present invention provides for the use ofSGA-56M or SGA-56Mv gene products, including SGA-56M or SGA-56Mv, and/orpeptide fragments thereof which can be used for the generation ofantibodies, in diagnostic assays, or for the identification of othercellular gene products involved in the development of cancer, such as,for example, breast cancer.

The amino acid sequences depicted in FIG. 7 and FIG. 8 representexamples of SGA-56M or SGA-56Mv gene products, i.e., SGA-56M (SEQ ID NO:5) or SGA-56Mv (SEQ ID NO: 6). The SGA-56M or SGA-56Mv gene products,sometimes referred to herein as an “SGA-56M or SGA-56Mv proteins” or“SGA-56M or SGA-56Mv polypeptides,” may additionally include those geneproducts encoded by the SGA-56M or SGA-56Mv gene sequences described inSection 5.1, above.

In addition, SGA-56M or SGA-56Mv derivatives may include proteins thathave conservative amino acid substitution(s) and/or display a functionalactivity of an SGA-56M or SGA-56Mv gene product, including but notlimited to SGA-56M or SGA-56Mv. Such a derivative may contain deletions,additions or substitutions of amino acid residues within the amino acidsequence encoded by the SGA-56M or SGA-56Mv gene sequences described,above, in Section 5.1, but which result in a conservative change, thusproducing a functionally equivalent SGA-56M or SGA-56Mv gene product.

In a specific embodiment, the invention provides a functionallyequivalent protein that exhibits a substantially similar in vivoactivity as an endogenous SGA-56M or SGA-56Mv gene product encoded by anSGA-56M or SGA-56Mv gene sequence described in Section 5.1, above. An invivo activity of the SGA-56M or SGA-56Mv gene product can be exhibitedby, for example, preneoplastic and/or neoplastic transformation of acell upon overexpression of the gene product, such as for example, mayoccur in the onset and progression and metastasis of breast cancer.

An SGA-56M or SGA-56Mv gene product sequence preferably comprises anamino acid sequence that exhibits at least about 65% sequence similarityto SGA-56M or SGA-56Mv, more preferably exhibits at least 70% sequencesimilarity to SGA-56M or SGA-56Mv, yet more preferably exhibits at leastabout 75% sequence similarity to SGA-56M or SGA-56Mv. In otherembodiments, the SGA-56M or SGA-56Mv gene product sequence preferablycomprises an amino acid sequence that exhibits at least 85% sequencesimilarity to SGA-56M or SGA-56Mv, yet more preferably exhibits at least90% sequence similarity to SGA-56M or SGA-56Mv, and most preferablyexhibits at least about 95% sequence similarity to SGA-56M or SGA-56Mv.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc Natl AcadSci. 87:2264-2268, modified as in Karlin and Altschul (1993) Proc NatlAcad Sci. 90:5873-5877. Such an algorithm is incorporated into theNBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol.215:403-410. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search that detects distant relationshipsbetween molecules (Id). When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated intothe ALIGN program (version 2.0) which is part of the GCG sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used. Additionalalgorithms for sequence analysis are known in the art and includeADVANCE and ADAM as described in Torellis and Robotti (1994) Comput.Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988)85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. For a further description of FASTA parameters, seehttp://bioweb.pasteur.fr/docs/man/man/fasta.1.html#sect2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.However, conservative substitutions should be considered in evaluatingsequences that have a low percent identity with the SGA-56M or SGA-56Mvsequences disclosed herein.

In a specific embodiment, molecules or protein comprising at least 10,20, 30, 40, 50, 75, 100, or 200 amino acids of SEQ ID NO: 5 or SEQ IDNO: 6 are used in the present invention.

5.2.1 Fusion Proteins

SGA-56M or SGA-56Mv gene products can also include fusion proteinscomprising an SGA-56M or SGA-56Mv gene product sequence as describedabove operatively linked or associated to a heterologous, component,e.g., peptide for use in the methods of the invention. Heterologouscomponents can include, but are not limited to sequences that facilitateisolation and purification of fusion protein, or label components.Heterologous components can also include sequences that confer stabilityto the SGA-56M or SGA-56Mv gene product or target the gene product to,for example, a particular tissue or cell type. Such isolation, labeling,and targeting components are well known to those of skill in the art.

The present invention encompasses the use of fusion proteins comprisingthe protein or fragment thereof encoded for by the SGA-56M or SGA-56Mvgene open reading frames SEQ ID NO: 2 or SEQ ID NO: 4 and a heterologouspolypeptide (i.e., an unrelated polypeptide or fragment thereof,preferably at least 10 to 100 amino acids of the polypeptide). Thefusion can be direct, but may occur through linker sequences. Theheterologous polypeptide may be fused to the N-terminus or C-terminus ofan SGA-56M or SGA-56Mv gene product.

A fusion protein can comprise an SGA-56M or SGA-56Mv gene product fusedto a signal sequence at its N-terminus. Various signal sequences arecommercially available. Eukaryotic heterologous signal sequencesinclude, but are not limited to, the secretory sequences of melittin andhuman placental alkaline phosphatase (Stratagene; La Jolla, Calif.).Prokaryotic heterologous signal sequences useful in the methods of theinvention include, but are not limited to, the phoA secretory signal(Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) and the protein A secretory signal (PharmaciaBiotech; Piscataway, N.J.).

The SGA-56M or SGA-56Mv protein (SEQ ID NO: 5 or SEQ ID NO: 6) orfragment thereof encoded by the SGA-56M or SGA-56Mv open reading framesSEQ ID NO: 2 or SEQ ID NO: 4, respectively, can be fused to tagsequences, e.g., a hexa-histidine peptide, such as the tag provided in apQE vector (QIAGEN, Inc., Chatsworth, Calif., 91311), among others, manyof which are commercially available for use in the methods of theinvention. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci.USA, 86:821-824, for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other examples of peptide tags arethe hemagglutinin “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell, 37:767)and the “flag” tag (Knappik et al., 1994, Biotechniques, 17(4):754-761).These tags are especially useful for purification of recombinantlyproduced polypeptides of the invention.

A fusion protein may readily be purified utilizing an antibodyspecific/selective for the fusion protein being expressed. For example,a system described by Janknecht et al. allows for the ready purificationof non-denatured fusion proteins expressed in human cell lines(Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972). In thissystem, the gene of interest is subcloned into a vaccinia recombinationplasmid such that the open reading frame of the gene is translationallyfused to an amino-terminal tag consisting of six histidine residues.Extracts from cells infected with recombinant vaccinia virus are loadedonto Ni²⁺•nitriloacetic acid-agarose columns and histidine-taggedproteins are selectively eluted with imidazole-containing buffers.

An affinity label may be fused, for example, at either the amino orcarboxyl terminal of the protein or fragment thereof encoded by anSGA-56M or SGA-56Mv open reading frame to generate a fusion protein foruse in the methods of the invention. The precise site at which a fusionis made in the carboxyl terminal, for example, is not critical. Theoptimal site can be determined by routine experimentation.

A variety of affinity labels known in the art may be used, such as, butnot limited to, the immunoglobulin constant regions, (Petty, 1996,Metal-chelate affinity chromatography, in Current Protocols in MolecularBiology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & WileyInterscience), glutathione S-transferase (GST; Smith, 1993, Methods Mol.Cell Bio. 4:220-229), the E. coli maltose binding protein (Guan et al.,1987, Gene 67:21-30), and various cellulose binding domains (U.S. Pat.Nos. 5,496,934; 5,202,247; 5,137,819; Tomme et al., 1994, Protein Eng.7:117-123), etc. Other affinity labels may impart fluorescent propertiesto an SGA-56M or SGA-56Mv gene product, e.g., green fluorescent proteinand the like. Other affinity labels are recognized by specific bindingpartners and thus facilitate isolation by affinity binding to thebinding partner that can be immobilized onto a solid support. Someaffinity labels may afford the SGA-56M or SGA-56Mv gene product novelstructural properties, such as the ability to form multimers. Theseaffinity labels are usually derived from proteins that normally exist ashomopolymers. Affinity labels such as the extracellular domains of CD8(Shine et al., 1988, J. Exp. Med. 168:1993-2005), or CD28 (Lee et al.,1990, J. Immunol. 145:344-352), or fragments of the immunoglobulinmolecule containing sites for interchain disulfide bonds, could lead tothe formation of multimers.

As will be appreciated by those skilled in the art, many methods can beused to obtain the coding region of the above-mentioned affinity labels,including but not limited to, DNA cloning, DNA amplification, andsynthetic methods. Some of the affinity labels and reagents for theirdetection and isolation are available commercially.

A preferred affinity label is a non-variable portion of theimmunoglobulin molecule. Typically, such portions comprise at least afunctionally operative CH2 and CH3 domain of the constant region of animmunoglobulin heavy chain. Fusions are also made using the carboxylterminus of the Fc portion of a constant domain, or a region immediatelyamino-terminal to the CH1 of the heavy or light chain. Suitableimmunoglobulin-based affinity label may be obtained from IgG-1, -2, -3,or -4 subtypes, IgA, IgE, IgD, or IgM, but preferably IgG1. Preferably,a human immunoglobulin is used when the SGA-56M or SGA-56Mv gene productis intended for in vivo use for humans. Many DNA encoding immunoglobulinlight or heavy chain constant regions are known or readily availablefrom cDNA libraries. See, for example, Adams et al., Biochemistry, 1980,19:2711-2719; Gough et al., 1980, Biochemistry, 19:2702-2710; Dolby etal., 1980, Proc. Natl. Acad. Sci. U.S.A., 77:6027-6031; Rice et al.,1982, Proc. Natl. Acad Sci U.S.A., 79:7862-7865; Falkner et al., 1982,Nature, 298:286-288; and Morrison et al., 1984, Ann. Rev. Immunol,2:239-256. Because many immunological reagents and labeling systems areavailable for the detection of immunoglobulins, the SGA-56M or SGA-56Mvgene product-Ig fusion protein can readily be detected and quantified bya variety of immunological techniques known in the art, such as the useof enzyme-linked immunosorbent assay (ELISA), immunoprecipitation,fluorescence activated cell sorting (FACS), etc. Similarly, if theaffinity label is an epitope with readily available antibodies, suchreagents can be used with the techniques mentioned above to detect,quantitate, and isolate the SGA-56M or SGA-56Mv gene product containingthe affinity label. In many instances, there is no need to developspecific or selective antibodies to the SGA-56M or SGA-56Mv geneproduct.

A fusion protein can comprise an SGA-56M or SGA-56Mv gene product fusedto the Fc domain of an immunoglobulin molecule or a fragment thereof foruse in the methods of the invention. A fusion protein can also comprisean SGA-56M or SGA-56Mv gene product fused to the CH2 and/or CH3 regionof the Fc domain of an immunoglobulin molecule. Furthermore, a fusionprotein can comprise an SGA-56M or SGA-56Mv gene product fused to theCH2, CH3, and hinge regions of the Fc domain of an immunoglobulinmolecule (see Bowen et al., 1996, J. Immunol. 156:44249). This hingeregion contains three cysteine residues that are normally involved indisulfide bonding with other cysteines in the Ig molecule. Since none ofthe cysteines are required for the peptide to function as a tag, one ormore of these cysteine residues may optionally be substituted by anotheramino acid residue, such as for example, serine.

Various leader sequences known in the art can be used for the efficientsecretion of the SGA-56M or SGA-56Mv gene product from bacterial andmammalian cells (von Heijne, 1985, J. Mol. Biol. 184:99-105). Leaderpeptides are selected based on the intended host cell, and may includebacterial, yeast, viral, animal, and mammalian sequences. For example,the herpes virus glycoprotein D leader peptide is suitable for use in avariety of mammalian cells. A preferred leader peptide for use inmammalian cells can be obtained from the V-J2-C region of the mouseimmunoglobulin kappa chain (Bernard et al., 1981, Proc. Natl. Acad. Sci.78:5812-5816). Preferred leader sequences for targeting SGA-56M orSGA-56Mv gene product expression in bacterial cells include, but are notlimited to, the leader sequences of the E. coli proteins OmpA (Hobom etal., 1995, Dev. Biol. Stand. 84:255-262), Pho A (Oka et al., 1985, Proc.Natl. Acad. Sci 82:7212-16), OmpT (Johnson et al., 1996, ProteinExpression 7:104-113), LamB and OmpF (Hoffman & Wright, 1985, Proc.Natl. Acad. Sci. USA 82:5107-5111), β-lactamase (Kadonaga et al., 1984,J. Biol. Chem. 259:2149-54), enterotoxins (Morioka-Fujimoto et al.,1991, J. Biol. Chem. 266:1728-32), Staphylococcus aureus protein A(Abrahmsen et al., 1986, Nucleic Acids Res. 14:7487-7500), and the B.subtilis endoglucanase (Lo et al., Appl. Environ. Microbiol.54:2287-2292), as well as artificial and synthetic signal sequences(MacIntyre et al., 1990, Mol. Gen. Genet. 221:466-74; Kaiser et al.,1987, Science, 235:312-317).

A fusion protein can comprise an SGA-56M or SGA-56Mv gene product and acell permeable peptide, which facilitates the transport of a protein orpolypeptide across the plasma membrane for use in the methods of theinvention. Examples of cell permeable peptides include, but are notlimited to, peptides derived from hepatitis B virus surface antigens(e.g., the PreS2-domain of hepatitis B virus surface antigens), herpessimplex virus VP22, antennapaedia, 6H, 6K, and 6R. See, e.g., Oess etal., 2000, Gene Ther. 7:750-758, DeRossi et al., 1998, Trends Cell Biol8(2):84-7, and Hawiger, 1997, J. Curr Opin Immunol 9(2):189-94.

Fusion proteins can be produced by standard recombinant DNA techniquesor by protein synthetic techniques, e.g., by use of a peptidesynthesizer. For example, a nucleic acid molecule encoding a fusionprotein can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Current Protocols in Molecular Biology, Ausubel etal., eds., John Wiley & Sons, 1992).

The nucleotide sequence coding for a fusion protein can be inserted intoan appropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. The expression of a fusion protein may beregulated by a constitutive, inducible or tissue-specific, or selectivepromoter. It will be understood by the skilled artisan that fusionproteins, which can facilitate solubility and/or expression, or canincrease the in vivo half-life of the protein or fragment thereofencoded by SGA-56M (SEQ ID NO: 2) or SGA-56Mv (SEQ ID NO: 4) and thusare useful in the methods of the invention. The SGA-56M or SGA-56Mv geneproducts or peptide fragments thereof, or fusion proteins can be used inany assay that detects or measures SGA-56M or SGA-56Mv gene products orin the calibration and standardization of such assay.

The methods of the invention encompass the use of SGA-56M or SGA-56Mvgene products or peptide fragments thereof, which may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the SGA-56M or SGA-56Mv gene polypeptides andpeptides of the invention by expressing nucleic acid containing SGA-56Mor SGA-56Mv gene sequences are described herein. Methods that are wellknown to those skilled in the art can be used to construct expressionvectors containing SGA-56M or SGA-56Mv gene product coding sequences(including but not limited to SEQ ID NO: 2 or SEQ ID NO: 4 andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra. Alternatively, RNA capable of encodingSGA-56M or SGA-56Mv gene product sequences may be chemically synthesizedusing, for example, synthesizers (see e.g., the techniques described inOligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford).

5.2.2 Expression Systems

A variety of host-expression vector systems may be utilized to expressthe SGA-56M or SGA-56Mv gene coding sequences for use in the methods ofthe invention. Such host-expression systems represent vehicles by whichthe coding sequences of interest may be produced and subsequentlypurified, but also represent cells which may, when transformed ortransfected with the appropriate nucleotide coding sequences, exhibitthe SGA-56M or SGA-56Mv gene product of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing SGA-56M orSGA-56Mv gene product coding sequences; yeast (e.g., Saccharomyces,Pichia) transformed with recombinant yeast expression vectors containingthe SGA-56M or SGA-56Mv gene product coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the SGA-56M or SGA-56Mv gene product codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; or tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing SGA-56M or SGA-56Mv gene product codingsequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the SGA-56Mor SGA-56Mv gene product being expressed. For example, when a largequantity of such a protein is to be produced, for the generation ofpharmaceutical compositions of SGA-56M or SGA-56Mv protein or forraising antibodies to SGA-56M or SGA-56Mv protein, vectors that directthe expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include, but are notlimited, to the E. coli expression vector pUR278 (Ruther et al., 1983,EMBO J. 2:1791), in which the SGA-56M or SGA-56Mv gene product codingsequence may be ligated individually into the vector in frame with thelac Z coding region so that a fusion protein is produced; pIN vectors(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101; Van Heeke &Schuster, 1989, J. Biol. Chem. 264:5503); and the like. pGEX vectors mayalso be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to a matrix of glutathione-agarose beads followed by elution inthe presence of free glutathione. The pGEX vectors are designed toinclude, e.g., thrombin or factor Xa protease cleavage sites so that thecloned target gene product can be released from the GST moiety.

In an insect system, Autographa califonica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The SGA-56M or SGA-56Mv gene codingsequence may be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of SGA-56M or SGA-56Mv gene coding sequence will result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat coded forby the polyhedrin gene). These recombinant viruses are then used toinfect Spodoptera frugiperda cells in which the inserted gene isexpressed (e.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S.Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the SGA-56M or SGA-56Mv gene coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing SGA-56M or SGA-56Mv gene product ininfected hosts. (See, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci.USA 81:3655). Specific initiation signals may also be required forefficient translation of inserted SGA-56M or SGA-56Mv gene productcoding sequences. These signals include the ATG initiation codon andadjacent sequences. In cases where an entire SGA-56M or SGA-56Mv gene,including its own initiation codon and adjacent sequences, is insertedinto the appropriate expression vector, no additional translationalcontrol signals may be needed. However, in cases where only a portion ofthe SGA-56M or SGA-56Mv gene coding sequence is inserted, exogenoustranslational control signals, including, perhaps, the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (See Bittner et al., 1987, Methods inEnzymol. 153:516).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB26, BT20 and T47D, and normal mammary glandcell lines such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theSGA-56M or SGA-56Mv gene product may be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and subsequentlyswitched to a selective media. The selectable marker in the recombinantplasmid confers the ability to grow in selective conditions. Cells thathave stably integrated the plasmid into their chromosomes grow to formfoci that in turn can be cloned and expanded into cell lines. Thismethod may advantageously be used to engineer cell lines that expressthe SGA-56M or SGA-56Mv gene product. Such engineered cell lines may beparticularly useful in screening and identifying compounds that affectthe endogenous activity of a SGA-56M and/or SGA-56Mv gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

5.2.3 SGA-56M or SGA-56Mv Transgenic Animals

The SGA-56M or SGA-56Mv gene products can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, guinea pigs, sheep, pigs, micro-pigs, goats,and non-human primates, e.g., baboons, monkeys, and chimpanzees may beused to generate SGA-56M or SGA-56Mv transgenic animals.

Transgenic animals that over- or mis-express an SGA-56M or SGA-56Mv geneproduct may be used in any of the methods of the invention. For exampletransgenic animals may be used to study the in vivo effects of enhancedexpression levels of SGA-56M or SGA-56Mv and the onset, diagnosis orprognosis of cancer. Transgenic animals are useful for screeningantagonists or agonists of SGA-56M or SGA-56Mv expression and/oractivity. Transgenic animals may also be used to screen the in vivoeffects of anti-sense or ribozyme therapeutic molecules in the treatmentof cancer. Transgenic animals could be used to screen for methods ofvaccinating against cancer using an SGA-56M or SGA-56Mv gene product ora portion thereof.

Further, SGA-56M or SGA-56Mv knock out animals are also useful in themethods of the invention. For example, animals with disruptions in onlySGA-56M or both SGA-56M and SGA-56Mv can be useful in assessing therelative contribution of each of these gene products to the cancerstate, as well as assessing the positive effect of a cancer therapeuticcandidate.

For over- or mis-expression of an SGA-56M or SGA-56Mv gene product, anytechnique known in the art may be used to introduce the SGA-56M orSGA-56Mv gene product into animals to produce the founder lines oftransgenic animals. Such techniques include, but are not limited topronuclear microinjection (Hoppe and Wagner, 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148); gene targetingin embryonic stem cells (Thompson et al., 1989, Cell 56:313);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717); etc.For a review of such techniques, see Gordon, 1989, Transgenic Animals,Intl. Rev. Cytol. 115:171.

The methods of the invention provide for the use of transgenic animalsthat carry the SGA-56M or SGA-56Mv transgene in all their cells, as wellas animals which carry the transgene in some, but not all their cells,i.e., mosaic animals.

The transgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko etal., 1992, Proc. Natl. Acad. Sci. USA 89:6232). The regulatory sequencesrequired for such a cell-type specific activation will depend upon theparticular cell type of interest, and will be apparent to those of skillin the art.

When it is desired that the SGA-56M/SGA-56Mv transgene be integratedinto the chromosomal site of the endogenous SGA-56M/SGA-56Mv gene, forexample to disrupt the expression of SGA-56M or both SGA-56M andSGA-56Mv, gene targeting is preferred. Briefly, when such a technique isto be utilized, vectors containing some nucleotide sequences homologousto the endogenous SGA-56M/SGA-56Mv gene are designed for the purpose ofintegrating, via homologous recombination with chromosomal sequences,into and partially or wholly disrupting the function of the nucleotidesequence of the endogenous SGA-56M/SGA-56Mv gene. The transgene may alsobe selectively introduced into a particular cell type, thus inactivatingthe endogenous SGA-56M/SGA-56Mv gene in only that cell type, byfollowing, for example, the teaching of Gu et al. (Gu et al., 1994,Science 265:103). The regulatory sequences required for such a cell-typespecific inactivation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art. It will beappreciated by a skilled artisan that the full length SGA-56M gene maybe targeted for specific modulation using such techniques by targetingthe region which is present in SGA-56M, but absent from SGA-56Mv, forhomologous recombination.

Methods for the production of single-copy transgenic animals with chosensites of integration are also well known to those of skill in the art.See, for example, Bronson et al. (Bronson, S. K. et al., 1996, Proc.Natl. Acad. Sci. USA 93:9067).

Once transgenic animals have been generated, expression of therecombinant SGA-56M/SGA-56Mv gene may be assayed utilizing standardtechniques. Initial screening may be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues to assay whetherintegration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals mayalso be assessed using techniques which include but are not limited toNorthern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-PCR. Samples of SGA-56M or SGA-56Mvgene-expressing tissue, may also be evaluated immunocytochemically usingantibodies specific/selective for the SGA-56M or SGA-56Mv gene product.

5.3. Antibodies to SGA-56M or SGA-56Mv Gene Products

The methods of the present invention encompass the use of antibodies orfragments thereof capable of specifically or selectively recognizing oneor more SGA-56M or SGA-56Mv gene product epitopes or epitopes ofconserved variants or peptide fragments of the SGA-56M or SGA-56Mv geneproducts. Such antibodies may include, but are not limited to,polyclonal antibodies, monoclonal antibodies (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, Fv fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above.

Such antibodies may be used, for example, in the detection of an SGA-56Mor SGA-56Mv gene product in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal levels of SGA-56M or SGA-56Mv geneproducts, and/or for the presence of abnormal variants of the such geneproducts. Such antibodies may also be included as a reagent in a kit foruse in a diagnostic and/or prognostic technique. Such antibodies mayalso be utilized in conjunction with, for example, compound screeningmethods, as described, below, in Section 5.5, for the evaluation of theeffect of test compounds on SGA-56M or SGA-56Mv gene product levelsand/or activity. Additionally, such antibodies can be used inconjunction with the gene therapy techniques described, below, inSection 5.6.4, to, for example, to evaluate the normal and/or engineeredSGA-56M or SGA-56Mv-expressing cells prior to their introduction intothe patient.

Antibodies to the SGA-56M or SGA-56Mv gene product may additionally beused in a method for the inhibition of SGA-56M or SGA-56Mv gene productactivity. Thus, such antibodies may, therefore, be utilized as part ofcancer treatment methods.

Described herein are methods for the production of antibodies orfragments thereof. Any of such antibodies or fragments thereof may beproduced by standard immunological methods or by recombinant expressionof nucleic acid molecules encoding the antibody or fragments thereof inan appropriate host organism.

For the production of antibodies against an SGA-56M or SGA-56Mv geneproduct, various host animals may be immunized by injection with anSGA-56M or SGA-56Mv gene product, or a portion thereof. Such hostanimals may include but are not limited to rabbits, mice, and rats, toname but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as an SGA-56M or SGA-56Mv gene product, or an antigenic functionalderivative thereof. For the production of polyclonal antibodies, hostanimals such as those described above, may be immunized by injectionwith SGA-56M or SGA-56Mv gene product supplemented with adjuvants asalso described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci USA80:2026), and the EBV-hybridoma technique (Cole et al., 1985, MonoclonalAntibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77). Suchantibodies may be of any immunoglobulin class including IgG, IgM, IgE,IgA, IgD and any subclass thereof. The hybridoma producing the mAb ofthis invention may be cultivated in vitro or in vivo. Production of hightiters of mAbs in vivo makes this the presently preferred method ofproduction.

Techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, Proc. Natl. Acad. Sci., 81, 6851-6855; Neubergeret al., 1984, Nature 312, 604-608; Takeda et al., 1985, Nature 314,452-454) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region.(See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al.,U.S. Pat. No. 5,816,397). The invention thus contemplates chimericantibodies that are specific/selective for an SGA-56M or SGA-56Mv geneproduct.

Examples of techniques that have been developed for the production ofhumanized antibodies are known in the art. (See, e.g., Queen, U.S. Pat.No. 5,585,089 and Winter, U.S. Pat. No. 5,225,539) An immunoglobulinlight or heavy chain variable region consists of a “framework” regioninterrupted by three hypervariable regions, referred to ascomplementarity-determining regions (CDRs). The extent of the frameworkregion and CDRs have been precisely defined (see, “Sequences of Proteinsof Immunological Interest”, Kabat, E. et al., U.S. Department of Healthand Human Services (1983). Briefly, humanized antibodies are antibodymolecules from non-human species having one or more CDRs from thenon-human species and framework regions from a human immunoglobulinmolecule. The invention includes the use of humanized antibodies thatare specific/selective for an SGA-56M and/or SGA-56Mv gene product inthe methods of the invention.

The method of the invention encompasses the use of an antibody orderivative thereof comprising a heavy or light chain variable domain,said variable domain comprising (a) a set of threecomplementarity-determining regions (CDRs), in which said set of CDRsare from a monoclonal antibody to a gene product encoded by an SGA-56Mnucleic acid sequence (SEQ ID NO: 2) or SGA-56Mv nucleic acid sequence(SEQ ID NO: 4), and (b) a set of four framework regions, in which saidset of framework regions differs from the set of framework regions ofthe SGA-56M and/or SGA-56Mv specific monoclonal antibody, and in whichsaid antibody or derivative thereof immunospecifically binds to the geneproduct encoded for by the SGA-56M or SGA-56Mv gene sequence.Preferably, the set of framework regions is from a human monoclonalantibody, e.g., a human monoclonal antibody that does not bind SGA-56Mor SGA-56Mv.

Phage display technology can be used to increase the affinity of anantibody to an SGA-56M or SGA-56Mv gene product. This technique isuseful for obtaining high affinity antibodies to an SGA-56M or SGA-56Mvgene product useful for the diagnosis and/or prognosis of a subject withcancer. The technology, referred to as affinity maturation, employsmutagenesis or CDR walking and re-selection using the SGA-56M orSGA-56Mv antigen to identify antibodies that bind with higher affinityto the antigen when compared with the initial or parental antibody (see,e.g., Glaser et al., 1992, J. Immunology 149:3903). Mutagenizing entirecodons rather than single nucleotides results in a semi-randomizedrepertoire of amino acid mutations. Libraries can be constructedconsisting of a pool of variant clones each of which differs by a singleamino acid alteration in a single CDR and which contain variantsrepresenting each possible amino acid substitution for each CDR residue.Mutants with increased binding affinity for the antigen can be screenedby contact with the immobilized mutants containing labeled antigen. Anyscreening method known in the art can be used to identify mutantantibodies with increased avidity to the antigen (e.g., ELISA) (See Wuet al., 1998, Proc Natl. Acad. Sci. USA 95:6037; Yelton et al., 1995, J.Immunology 155:1994). CDR walking may also be used to randomize thelight chain (See Schier et al., 1996, J. Mol. Bio. 263:551).

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Hustonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; and Ward et al., 1989,Nature 334:544) can be adapted to produce single chain antibodiesagainst SGA-56M or SGA-56Mv gene products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.Techniques for the assembly of functional Fv fragments in E. coli mayalso be used (Skerra et al., 1988, Science 242:1038).

The methods of the invention include using an antibody to an SGA-56M orSGA-56Mv polypeptide, peptide or other derivative, or analog thereofthat is a bispecific antibody (see generally, e.g., Fanger and Drakeman,1995, Drug News and Perspectives 8:133-137). Bispecific antibodies canbe used for example to treat and/or prevent cancer in a subject thatexpresses elevated levels of an SGA-56M or SGA-56Mv gene product Such abispecific antibody is genetically engineered to recognize both (1) anepitope and (2) one of a variety of “trigger” molecules, e.g., Fcreceptors on myeloid cells, and CD3 and CD2 on T-cells, that have beenidentified as capable of inducing a cytotoxic T-cell to destroy aparticular target. Such bispecific antibodies can be prepared either bychemical conjugation, hybridoma, or recombinant molecular biologytechniques known to the skilled artisan.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: F(ab′)₂ fragments which can be produced by pepsin digestionof the antibody molecule and Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively,Fab expression libraries may be constructed (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity.

5.4. Uses of the SGA-56M and SGA-56Mv Gene, Gene Products, andAntibodies

In various embodiments, the present invention provides various uses ofthe SGA-56M or SGA-56Mv gene, the SGA-56M or SGA-56Mv polypeptides andpeptide fragments thereof, and of antibodies directed against theSGA-56M or SGA-56Mv polypeptides and peptide fragments. Such usesinclude, for example, prognostic and diagnostic evaluation of cancer,and the identification of subjects with a predisposition to a cancer, asdescribed, herein below. The invention also includes methods of treatingand/or preventing cancer. The invention includes methods of vaccinatingagainst cancer. The methods of the invention can be used for thetreatment, prevention, vaccination, diagnosis, staging and/or prognosisof any cancer, or tumor, for example, but not limited to, any of thetumors or cancers listed below in Table 1.

Malignancies and related disorders, cells of which type can be tested invitro (and/or in vivo), and upon observing the appropriate assay result,treated according to the methods of the present invention, include butare not limited to those listed in Table 1 (for a review of suchdisorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B. LippincottCo., Philadelphia). TABLE 1 MALIGNANCIES AND RELATED DISORDERS Leukemia  acute leukemia     acute lymphocytic leukemia     acute myelocyticleukemia       myeloblastic       promyelocytic       myelomonocytic      monocytic       erythroleukemia   chronic leukemia     chronicmyelocytic (granulocytic) leukemia     chronic lymphocytic leukemiaPolycythemia vera Lymphoma   Hodgkin's disease   non-Hodgkin's diseaseMultiple myeloma Waldenström's macroglobulinemia Heavy chain diseaseSolid tumors   sarcomas and carcinomas     fibrosarcoma     myxosarcoma    liposarcoma     chondrosarcoma     osteogenic sarcoma     chordoma    angiosarcoma     endotheliosarcoma     lymphangiosarcoma    lymphangioendotheliosarcoma     synovioma     mesothelioma    Ewing's tumor     leiomyosarcoma     rhabdomyosarcoma     coloncarcinoma     pancreatic cancer     breast cancer     ovarian cancer    prostate cancer     squamous cell carcinoma     basal cell carcinoma    adenocarcinoma     sweat gland carcinoma     sebaceous glandcarcinoma     papillary carcinoma     papillary adenocarcinomas    cystadenocarcinoma     medullary carcinoma     bronchogeniccarcinoma     renal cell carcinoma     hepatoma     bile duct carcinoma    choriocarcinoma     seminoma     embryonal carcinoma     Wilms'tumor     cervical cancer     testicular tumor     lung carcinoma    small cell lung carcinoma     bladder carcinoma     epithelialcarcinoma     glioma     astrocytoma     medulloblastoma    craniopharyngioma     ependymoma     pinealoma     hemangioblastoma    acoustic neuroma     oligodendroglioma     menangioma     melanoma    neuroblastoma     retinoblastoma

In a preferred embodiment the methods of the invention are directed atdiagnosis, prognosis, treatment and prevention of breast cancer. Inother embodiments, the cancer is ovarian cancer, skin cancer, or cancerof the lymphoid system

The invention further provides for screening assays to identifyantagonists or agonists of the SGA56M or SGA-56Mv gene or gene product.Thus, the invention relates to methods to identify molecules thatup-regulate or down-regulate expression of the SGA-56M or SGA-56Mv gene.

The nucleic acid molecules, proteins, protein homologs, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) detection assays (e.g., chromosomal mapping, tissuetyping); c) predictive medicine (e.g., diagnostic assays, prognosticassays, monitoring clinical trials, and pharmacogenomics); and d)methods of treatment (e.g., therapeutic and prophylactic). For example,an SGA-56M or SGA-56Mv gene product can be used to modulate (i) cellularproliferation; (ii) cellular differentiation; and/or (iii) cellularadhesion. Isolated nucleic acid molecules that encode the SGA-56M orSGA-56Mv gene or a fragment or an open reading frame thereof can be usedto express proteins (e.g., via a recombinant expression vector in a hostcell in gene therapy applications), to detect mRNA (e.g., in abiological sample) or a genetic lesion, and to modulate activity of anSGA-56M or SGA-56Mv polypeptide. In addition, an SGA-56M or SGA-56Mvgene product can be used to screen drugs or compounds which modulateactivity or expression of the SGA-56M or SGA-56Mv gene product as wellas to treat disorders characterized by insufficient or excessiveproduction of the SGA-56M or SGA-56Mv gene product or production of aform the SGA-56M or SGA-56Mv gene product which has decreased oraberrant activity compared to the wild type protein. In addition, theantibodies that specifically or selectively bind to an SGA-56M orSGA-56Mv gene product can be used to detect, isolate, and modulateactivity of the SGA-56M or SGA-56Mv gene product.

In one embodiment, the present invention provides a variety of methodsfor the diagnostic and prognostic evaluation of cancer, including breastcancer. Such methods may, for example, utilize reagents such as theSGA-56M or SGA-56Mv gene nucleotide sequences described in Sections 5.1,and antibodies directed against SGA-56M or SGA-56Mv gene products,including peptide fragments thereof, as described, above, in Section5.2. Specifically, such reagents may be used, for example, for: (1) thedetection of the presence of SGA-56M or SGA-56Mv gene mutations, or thedetection of either over- or under-expression of SGA-56M or SGA-56Mvgene mRNA, preneoplastic or neoplastic, relative to normal cells or thequalitative or quantitative detection of other allelic forms of SGA-56Mor SGA-56Mv transcripts which may correlate with breast cancer orsusceptibility toward neoplastic changes, and (2) the detection of anover-abundance of an SGA-56M or SGA-56Mv gene product relative to anon-diseased state or relative to a predetermined non-cancerous standardor the presence of a modified (e.g., less than full-length) SGA-56M orSGA-56Mv gene product which correlates with a neoplastic state or aprogression toward neoplasia or metastasis.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic test kits comprising at least one specific orselective SGA-56M or SGA-56Mv gene nucleic acid or anti-SGA-56M oranti-SGA-56Mv antibody reagent described herein, which may beconveniently used, e.g., in clinical settings or in home settings, todiagnose patients exhibiting preneoplastic or neoplastic abnormalities,and to screen and identify those individuals exhibiting a predispositionto such neoplastic changes.

Nucleic acid-based detection techniques are described, below, in Section5.4.1. Peptide detection techniques are described, below, in Section5.4.2.

5.4.1. Detection of SGA-56M or SGA-56Mv Gene Nucleic Acid Molecules

In a preferred embodiment, the invention involves methods to assessquantitative and qualitative aspects of SGA-56M or SGA-56Mv geneexpression. In one example the increased expression of an SGA-56M orSGA-56Mv gene or gene product indicates a predisposition for thedevelopment of cancer. Alternatively, enhanced expression levels of anSGA-56M or SGA-56Mv gene or gene product can indicate the presence ofcancer in a subject or the risk of metastasis of said cancer in saidsubject. Techniques well known in the art, e.g., quantitative orsemi-quantitative RT PCR or Northern blot, can be used to measureexpression levels of SGA-56M or SGA-56Mv. Methods that describe bothqualitative and quantitative aspects of SGA-56M or SGA-56Mv gene or geneproduct expression are described in detail in the examples infra. Themeasurement of SGA-56M or SGA-56Mv gene expression levels can includemeasuring naturally occurring SGA-56M or SGA-56Mv transcripts andvariants thereof as well as non-naturally occurring variants thereof,however for the diagnosis and/or prognosis of cancer in a subject theSGA-56M or SGA-56Mv gene product is preferably a naturally occurringSGA-56M or SGA-56Mv gene product or variant thereof. Thus, the inventionrelates to methods of diagnosing and/or predicting cancer in a subjectby measuring the expression of the SGA-56M or SGA-56Mv gene in asubject. For example an increased level of mRNA encoded by a SGA-56M orSGA-56Mv nucleic acid sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 3), orother gene product, as compared to a non-cancerous sample or anon-cancerous predetermined standard would indicate the presence ofcancer in said subject or the increased risk of developing cancer insaid subject.

In another example the increased level of mRNA encoded for by an SGA-56Mor SGA-56Mv nucleic acid sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 3),or other gene product, as compared to a non-cancerous sample or anon-cancerous predetermined standard would indicate the risk ofmetastasis in a cancer subject or the likelihood of a poor prognosis insaid subject.

In another example, RNA from a cell type or tissue known, or suspected,to express the SGA-56M or SGA-56Mv gene, such as breast cancer cells, orother types of cancer cells, including metastases, may be isolated andtested utilizing hybridization or PCR techniques as described, above.The isolated cells can be derived from cell culture or from a patient.The analysis of cells taken from culture may be a necessary step in theassessment of cells to be used as part of a cell-based gene therapytechnique or, alternatively, to test the effect of compounds on theexpression of the SGA-56M or SGA-56Mv gene. Such analyses may revealboth quantitative and qualitative aspects of the expression pattern ofthe SGA-56M or SGA-56Mv gene, including activation or inactivation ofSGA-56M or SGA-56Mv gene expression and presence of alternativelyspliced SGA-56M or SGA-56Mv transcripts.

In one embodiment of such a detection scheme, a cDNA molecule issynthesized from an RNA molecule of interest by reverse transcription.All or part of the resulting cDNA is then used as a template for anucleic acid amplification reaction, such as a PCR or the like. Thenucleic acid reagents used as synthesis initiation reagents (e.g.,primers) in the reverse transcription and nucleic acid amplificationsteps of this method are chosen from among the SGA-56M or SGA-56Mv genenucleic acid reagents described in Section 5.1. The preferred lengths ofsuch nucleic acid reagents are at least 9-30 nucleotides.

For detection of the amplified product, the nucleic acid amplificationmay be performed using radioactively or non-radioactively labelednucleotides. Alternatively, enough amplified product may be made suchthat the product may be visualized by standard ethidium bromide stainingor by utilizing any other suitable nucleic acid staining method.

RT-PCR techniques can be utilized to detect differences in SGA-56M orSGA-56Mv transcript size that may be due to normal or abnormalalternative splicing. Additionally, such techniques can be performedusing standard techniques to detect quantitative differences betweenlevels of SGA-56M or SGA-56Mv transcripts detected in normal individualsrelative to those individuals having cancer or exhibiting apredisposition toward neoplastic changes.

In the case where detection of particular alternatively spliced speciesis desired, appropriate primers and/or hybridization probes can be used,such that, in the absence of such a sequence, for example, noamplification products are generated. Alternatively, primer pairs may bechosen utilizing the sequence data depicted in FIG. 1 or FIG. 2 to yieldfragments of differing size depending on whether a particular exon ispresent or absent from the transcript of SGA-56M or SGA-56Mv beinganalyzed.

As an alternative to amplification techniques, standard Northernanalyses can be performed if a sufficient quantity of the appropriatecells can be obtained. The preferred length of a probe used in aNorthern analysis may, for example, be 9-50 nucleotides. Utilizing suchtechniques, quantitative as well as size related differences betweenSGA-56M or SGA-56Mv transcripts can also be detected.

Additionally, it is possible to perform such SGA-56M or SGA-56Mv geneexpression assays in situ, i.e., directly upon tissue sections (fixedand/or frozen) of patient tissue obtained from biopsies or resections,such that no nucleic acid purification is necessary. Nucleic acidreagents such as those described in Section 5.1 may be used as probesand/or primers for such in situ procedures (see, e.g., Nuovo, G. J.,1992, PCR In Situ Hybridization: Protocols And Applications, RavenPress, N.Y.).

Mutations or polymorphisms within the SGA-56M or SGA-56Mv gene can bedetected by utilizing a number of techniques. Nucleic acid from anynucleated cell can be used as the starting point for such assaytechniques, and may be isolated according to standard nucleic acidpreparation procedures that are well known to those of skill in the art.For the detection of SGA-56M or SGA-56Mv mutations, any nucleated cellcan be used as a starting source for genomic nucleic acid. For thedetection of SGA-56M or SGA-56Mv transcripts or SGA-56M or SGA-56Mv geneproducts, any cell type or tissue in which the SGA-56M or SGA-56Mv geneis expressed, such as, for example, breast cancer cells, includingmetastases, may be utilized.

Genomic DNA may be used in hybridization or amplification assays ofbiological samples to detect abnormalities involving SGA-56M or SGA-56Mvgene structure, including point mutations, insertions, deletions andchromosomal rearrangements. Such assays may include, but are not limitedto, direct sequencing (Wong, C. et al., 1987, Nature 330:384), singlestranded conformational polymorphism analyses (SSCP; Orita, M. et al.,1989, Proc. Natl. Acad. Sci. USA 86:2766), heteroduplex analysis (Keen,T. J. et al., 1991, Genomics 11: 199; Perry, D. J. & Carrell, R. W.,1992), denaturing gradient gel electrophoresis (DGGE; Myers, R. M. etal., 1985, Nucl. Acids Res. 13:3131), chemical mismatch cleavage(Cotton, R. G. et al., 1988, Proc. Natl. Acad. Sci. USA 85:4397) andoligonucleotide hybridization (Wallace, R. B. et al., 1981, Nucl. AcidsRes. 9:879; Lipshutz, R. J. et al., 1995, Biotechniques 19:442).

Diagnostic methods for the detection of SGA-56M or SGA-56Mv nucleic acidmolecules, in patient samples or other appropriate cell sources, mayinvolve the amplification of specific gene sequences, e.g., by thepolymerase chain reaction (PCR; See Mullis, K. B., 1987, U.S. Pat. No.4,683,202), followed by the analysis of the amplified molecules usingtechniques well known to those of skill in the art, such as, forexample, those listed above. Utilizing analysis techniques such asthese, the amplified sequences can be compared to those that would beexpected if the nucleic acid being amplified contained only normalcopies of the SGA-56M or SGA-56Mv gene in order to determine whether anSGA-56M or SGA-56Mv gene mutation exists.

Further, well-known genotyping techniques can be performed to typepolymorphisms that are in close proximity to mutations in the SGA-56M orSGA-56Mv gene itself. These polymorphisms can be used to identifyindividuals in families likely to carry mutations. If a polymorphismexhibits linkage disequilibrium with mutations in the SGA-56M orSGA-56Mv gene, it can also be used to identify individuals in thegeneral population likely to carry mutations. Polymorphisms that can beused in this way include restriction fragment length polymorphisms(RFLPs), which involve sequence variations in restriction enzyme targetsequences, single-nucleotide polymorphisms (SNPs) and simple sequencerepeat polymorphisms (SSLPs).

For example, Weber (U.S. Pat. No. 5,075,217) describes a DNA markerbased on length polymorphisms in blocks of (dC-dA)n-(dG-dT)n shorttandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocks isestimated to be 30,000-60,000 bp. Markers that are so closely spacedexhibit a high frequency of co-inheritance, and are extremely useful inthe identification of genetic mutations, such as, for example, mutationswithin the SGA-56M or SGA-56Mv gene, and the diagnosis of diseases anddisorders related to SGA-56M or SGA-56Mv mutations.

Also, Caskey et al. (U.S. Pat. No. 5,364,759), describe a DNA profilingassay for detecting short tri- and tetra-nucleotide repeat sequences.The process includes extracting the DNA of interest, such as the SGA-56Mor SGA-56Mv gene, amplifying the extracted DNA, and labeling the repeatsequences to form a genotypic map of the individual's DNA.

An SGA-56M or SGA-56Mv probe could be used to directly identify RFLPs.Additionally, an SGA-56M or SGA-56Mv probe or primers derived from theSGA-56M or SGA-56Mv sequence could be used to isolate genomic clonessuch as YACs, BACs, PACs, cosmids, phage or plasmids. The DNA containedin these clones can be screened for SNPs or SSLPs using standardhybridization or sequencing procedures.

Alternative diagnostic methods for the detection of SGA-56M or SGA-56Mvgene expression, SGA-56M or SGA-56Mv gene mutations or polymorphisms caninclude hybridization techniques which involve for example, contactingand incubating nucleic acids including recombinant DNA molecules, clonedgenes or degenerate variants thereof, obtained from a sample, e.g.,derived from a patient sample or other appropriate cellular source, withone or more labeled nucleic acid reagents including recombinant DNAmolecules, cloned genes or degenerate variants thereof, as described inSection 5.1, under conditions favorable for the specific or selectiveannealing of these reagents to their complementary sequences within theSGA-56M or SGA-56Mv gene. Preferably, the lengths of these nucleic acidreagents are at least 9 to 50 nucleotides. After incubation, allnon-annealed nucleic acids are removed from the nucleic acid: SGA-56M orSGA-56Mv molecule hybrid. The presence of nucleic acids that havehybridized, if any such molecules exist, is then detected. Using such adetection scheme, a nucleic acid from the cell type or tissue ofinterest can be immobilized, for example, to a solid support such as amembrane, or a plastic surface such as that on a microtiter plate orpolystyrene beads or to a glass surface such as a microscope slide. Inthis case, after incubation, non-annealed, labeled nucleic acid reagentsof the type described in Section 5.1 are easily removed. Detection ofthe remaining, annealed, labeled SGA-56M or SGA-56Mv nucleic acidreagents is accomplished using standard techniques well known in theart. The SGA-56M or SGA-56Mv gene sequences to which the nucleic acidreagents have annealed can be compared to the annealing pattern expectedfrom a normal SGA-56M or SGA-56Mv gene sequence in order to determinewhether an SGA-56M or SGA-56Mv gene mutation is present.

5.4.2. Detection of SGA-56M and SGA-56Mv Encoded Proteins

Detection of the SGA-56M or SGA-56Mv gene product includes the detectionof the proteins comprising SEQ ID NO: 5 or SEQ ID NO: 6. Detection ofelevated levels of SGA-56M or SGA-56Mv, compared to a non-canceroussample or a non-cancerous predetermined standard can indicate thepresence of cancer, or predisposition to developing cancer in a subject.Detection of elevated levels of said protein in a subject compared to anon-cancerous sample or a non-cancerous predetermined standard canindicate the likelihood of metastasis of a cancer in the subject, and/orpoor prognosis for the subject. The diagnosis and/or prognosis of cancerpertains to the detection of naturally occurring SGA-56M or SGA-56Mvpolypeptides in a subject Detection of an SGA-56M or SGA-56Mvpolypeptide can be by any method known in the art.

Antibodies directed against naturally occurring SGA-56M or SGA-56Mv, ornaturally occurring variants thereof or peptide fragments thereof, whichare discussed, above, in Section 5.2, may be used as diagnostics andprognostics, as described herein. Such diagnostic methods, may be usedto detect abnormalities in the level of SGA-56M or SGA-56Mv geneexpression, or abnormalities in the structure and/or temporal, tissue,cellular, or subcellular location of the SGA-56M or SGA-56Mv-encodedpolypeptide. Antibodies, or fragments of antibodies, such as thosedescribed herein, may be used to screen potentially therapeuticcompounds in vitro to determine their effects on SGA-56M or SGA-56Mvgene expression and SGA-56M or SGA-56Mv-encoded polypeptide production.The compounds that have beneficial effects on cancer, e.g., breastcancer can be identified and a therapeutically effective dosedetermined.

The tissue or cell type to be analyzed will generally include thosewhich are known, or suspected, to express the SGA-56M or SGA-56Mv gene,such as, for example, cancer cells including breast cancer cells,ovarian cancer cells, skin cancer cells, lymphoid cancer cells, andmetastatic forms thereof. The protein isolation methods employed hereinmay, for example, be such as those described in Harlow and Lane (Harlow,E. and Lane, D., 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). The isolated cellscan be derived from cell culture or from a patient. The analysis ofcells taken from culture may be a necessary step to test the effect ofcompounds on the expression of the SGA-56M or SGA-56Mv gene.

Preferred diagnostic methods for the detection of SGA-56M or SGA-56Mvgene products or conserved variants or peptide fragments thereof, mayinvolve, for example, immunoassays wherein the SGA-56M or SGA-56Mv geneproducts or conserved variants, including gene products which are theresult of alternatively spliced transcripts, or peptide fragments aredetected by their interaction with an anti-SGA-56M or anti-SGA-56Mv geneproduct specific antibody.

For example, antibodies, or fragments of antibodies, such as thosedescribed above in Section 5.3, useful in the present invention may beused to quantitatively or qualitatively detect the presence of SGA-56Mor SGA-56Mv-encoded polypeptides or naturally occurring variants orpeptide fragments thereof. The antibodies (or fragments thereof) usefulin the present invention may, additionally, be employed histologically,as in immunofluorescence or immunoelectron microscopy, for in situdetection of SGA-56M or SGA-56Mv gene products or conserved variants orpeptide fragments thereof. In situ detection may be accomplished byremoving a histological specimen from a subject, such as paraffinembedded sections of tissue, e.g., breast tissues, and applying theretoa labeled antibody of the present invention. The antibody (or fragment)is preferably applied by overlaying the labeled antibody (or fragment)onto a biological sample. Since the SGA-56M or SGA-56Mv gene product ispresent in the cytoplasm, it may be desirable to introduce the antibodyinside the cell, for example, by making the cell membrane permeable. TheSGA-56M or SGA-56Mv polypeptides may also be expressed on the cellsurface, thus cells can be directly labeled by applying antibodies thatare specific or selective for the SGA-56M or SGA-56Mv polypeptides orfragment thereof to the cell surface.

Through the use of such a procedure, it is possible to determine notonly the presence of the SGA-56M or SGA-56Mv gene product, or naturallyoccurring variants thereof or peptide fragments, but also itsdistribution in the examined tissue. Using the methods of the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays for SGA-56M or SGA-56Mv-encoded polypeptides or conservedvariants or peptide fragments thereof will typically comprise contactinga sample, such as a biological fluid, tissue or a tissue extract,freshly harvested cells, or lysates of cells which have been incubatedin cell culture, in the presence of an antibody that specifically orselectively binds to an SGA-56M or SGA-56Mv gene product, e.g., adetectably labeled antibody capable of identifying SGA-56M or SGA-56Mvpolypeptides or conserved variants or peptide fragments thereof, anddetecting the bound antibody by any of a number of techniques well-knownin the art (e.g., Western blot, ELISA, FACS).

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support that is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled antibody thatselectively or specifically binds to an SGA-56M or SGA-56Mv-encodedpolypeptide. The solid phase support may then be washed with the buffera second time to remove unbound antibody. The amount of bound label onsolid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The anti-SGA-56M or anti-SGA-56Mv antibody can be detectably labeled bylinking the same to an enzyme and using the labeled antibody in anenzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)”, 1978, Diagnostic Horizons 2:1, MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.); Voller, A. et al.,1978, J. Clin. Pathol. 31: 507-520; Butler, J. E., 1981, Meth. Enzymol.73:482; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, BocaRaton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay,Kgaku Shoin, Tokyo). The enzyme that is bound to the antibody will reactwith an appropriate substrate, preferably a chromogenic substrate, insuch a manner as to produce a chemical moiety that can be detected, forexample, by spectrophotometric or fluorimetric means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby calorimetric methods that employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect SGA-56M or SGA-56Mv-encodedpolyepeptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986). The radioactive isotope can be detected by such means asthe use of a gamma counter, a scintillation counter, or byautoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due to fluorescenceemission. Among the most commonly used fluorescent labeling compoundsare fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetiaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems wherein a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

In various embodiments, the present invention provides methods for themeasurement of SGA-56M or SGA-56Mv polypeptides, and the uses of suchmeasurements in clinical applications using SGA-56M or SGA-56Mv specificantibodies.

The measurement of SGA-56M or SGA-56Mv polyppeptides of the inventioncan be valuable in detecting and/or staging breast cancer and othercancers in a subject, in screening of breast cancer and other cancers ina population, in differential diagnosis of the physiological conditionof a subject, and in monitoring the effect of a therapeutic treatment ona subject.

The present invention also provides for detecting, diagnosing, orstaging of breast cancer and other cancers, or the monitoring oftreatment of breast cancer and other cancers by measuring the level ofexpression of an SGA-56M or SGA-56Mv polypeptide. In addition to theSGA-56M or SGA-56Mv polypeptide at least one other marker, such as areceptor or differentiation antigen can also be measured. For example,serum markers selected from, for example but not limited to,carcinoembryonic antigen (CEA), CA15-3, CA549, CAM26, M29, CA27.29 andMCA can be measured in combination with an SGA-56M or SGA-56Mvpolypeptide to detect, diagnose, stage, and/or monitor treatment ofbreast cancer and other cancers. In another embodiment, the prognosticindicator is the observed change in different marker levels relative toone another, rather than the absolute levels of the markers present atany one time. These measurements can also aid in predicting therapeuticoutcome and in evaluating and monitoring the overall disease status of asubject.

In a specific embodiment of the invention, soluble SGA-56M or SGA-56Mvpolypeptide alone or in combination with other markers can be measuredin any body fluid of the subject including but not limited to blood,serum, plasma, milk, urine, saliva, pleural effusions, synovial fluid,spinal fluid, tissue infiltrations and tumor infiltrates. In anotherembodiment an SGA-56M or SGA-56Mv polypeptide is measured in tissuesamples or cells directly. The present invention also contemplates a kitfor measuring the level of SGA-56M or SGA-56Mv expression in abiological sample and the use of said kit to diagnose a subject withcancer. Alternatively said kit could be used to determine the prognosisof a cancer patient or the risk of metastasis of said cancer.

Any of numerous immunoassays can be used in the practice of the methodsof the instant invention, such as those described in Section 5.4.2.Antibodies, or antibody fragments containing the binding domain, whichcan be employed include, but are not limited to, suitable antibodiesamong those in Section 5.3 and other antibodies known in the art orwhich can be obtained by procedures standard in the art such as thosedescribed in Section 5.3.

5.4.2.1 In Vivo Imaging Using Antibodies to an SGA-56M or SGA-56MvPolypeptide

Current diagnostic and therapeutic methods make use of antibodies totarget imaging agents or therapeutic substances, e.g., to tumors. Thus,labeled antibodies immunologically specific for an SGA-56M or SGA-56Mvpolypepeptide can be used in the methods of the invention for the invivo imaging, detection, and treatment of cancer in a subject.

Antibodies may be linked to chelators such as those described in U.S.Pat. No. 4,741,900 or U.S. Pat. No. 5,326,856. The antibody-chelatorcomplex may then be radiolabeled to provide an imaging agent fordiagnosis and/or treatment of disease. The antibodies may also be usedin the methods that are disclosed in U.S. Pat. No. 5,449,761 forcreating a radiolabeled antibody for use in imaging or radiotherapy.

In in vivo diagnostic applications, specific tissues or even specificcellular disorders, e.g., cancer, may be imaged by administration of asufficient amount of a labeled antibody using the methods of the instantinvention.

A wide variety of metal ions suitable for in vivo tissue imaging havebeen tested and utilized clinically. For imaging with radioisotopes, thefollowing characteristics are generally desirable: (a) low radiationdose to the patient; (b) high photon yield which permits a nuclearmedicine procedure to be performed in a short time period; (c) abilityto be produced in sufficient quantities; (d) acceptable cost; (e) simplepreparation for administration; and (f) no requirement that the patientbe sequestered subsequently. These characteristics generally translateinto the following: (a) the radiation exposure to the most criticalorgan is less than 5 rad; (b) a single image can be obtained withinseveral hours after infusion; (c) the radioisotope does not decay byemission of a particle; (d) the isotope can be readily detected; and (e)the half-life is less than four days (Lamb and Kramer, “CommercialProduction of Radioisotopes for Nuclear Medicine”, In Radiotracers ForMedical Applications, Vol. 1, Rayudu (Ed.), CRC Press, Inc., Boca Raton,pp. 17-62). Preferably, the metal is technetium-99m.

By way of illustration, the targets that one may image include any solidneoplasm, certain organs such as lymph nodes, parathyroids, spleen andkidney, sites of inflammation or infection (e.g., macrophages at suchsites), myocardial infarction or thromboses (neoantigenic determinantson fibrin or platelets), and the like evident to one of ordinary skillin the art. Furthermore, the neoplastic tissue may be present in bone,internal organs, connective tissue, or skin.

As is also apparent to one of ordinary skill in the art, one may use themethods of the present invention in in vivo therapeutics (e.g., usingradiotherapeutic metal complexes), especially after having diagnosed adiseased condition via the in vivo diagnostic method described above, orin in vitro diagnostic application (e.g., using a radiometal or afluorescent metal complex).

Accordingly, a method of diagnosing cancer by obtaining an image of aninternal region of a subject is contemplated in the instant inventionwhich comprises administering to a subject an effective amount of anantibody composition specific for an SGA-56M or SGA-56Mv polypeptideconjugated with a metal in which the metal is radioactive, and recordingthe scintigraphic image obtained from the decay of the radioactivemetal. Likewise, a method is contemplated of enhancing a magneticresonance image (MRI) of an internal region of a subject which comprisesadministering to a subject an effective amount of an antibodycomposition containing a metal in which the metal is paramagnetic, andrecording the MRI of an internal region of the subject.

Other methods are directed to enhancing a sonographic image of aninternal region of a subject comprising administering to a subject aneffective amount of an antibody composition containing a metal andrecording the sonographic image of an internal region of the subject. Inthis latter application, the metal is preferably any non-toxic heavymetal ion. A method of enhancing an X-ray image of an internal region ofa subject is also provided which comprises administering to a subject anantibody composition containing a metal, and recording the X-ray imageof an internal region of the subject. A radioactive, non-toxic heavymetal ion is preferred.

5.43. Detecting and Staging Cancer in a Subject

The methods of the present invention include measurement of a naturallyoccurring SGA-56M or SGA-56Mv polypeptide, or naturally occurringvariants thereof, or fragment thereof, soluble SGA-56M or SGA-56Mvpolypeptide or intracellular SGA-56M or SGA-56Mv polypeptides to detectbreast cancer or other cancers in a subject or to stage breast cancer orother cancers in a subject.

Staging refers to the grouping of patients according to the extent oftheir disease. Staging is useful in choosing treatment for individualpatients, estimating prognosis, and comparing the results of differenttreatment programs. Staging of breast cancer for example is performedinitially on a clinical basis, according to the physical examination andlaboratory radiologic evaluation. The most widely used clinical stagingsystem is the one adopted by the International Union against Cancer(UICC) and the American Joint Committee on Cancer (AJCC) Staging and EndResults Reporting. It is based on the tumor-nodes-metastases (TNM)system as detailed in the 1988 Manual for Staging of Cancer. Breastcancer diseases or conditions that may be detected and/or staged in asubject according to the present invention include but are not limitedto those listed in Table 2. TABLE 2 STAGING OF BREAST CANCER T PRIMARYTUMORS TX Primary tumor cannot be assessed T0 No evidence of primarytumor Tis Carcinoma in situ: intraductal carcinoma, lobular carcinoma,or Paget's disease with no tumor T1 Tumor 2 cm or less in its greatestdimension a. 0.5 cm or less in greatest dimension b. Larger than 0.5 cm,but not larger than 1 cm in greatest dimension c. Larger than 1 cm, butnot larger than 2 cm in greatest dimension T2 Tumor more than 2 cm butnot more than 5 cm in greatest dimension T3 Tumor more than 5 cm in itsgreatest dimension T4 Tumor of any size with direct extension to chestwall or to skin. Chest wall includes ribs, intercostal muscles, andserratus anterior muscle, but not pectoral muscle. a. Extension to chestwall b. Edema (including peau d'orange), ulceration of the skin of thebreast, or satellite skin nodules confined to the same breast c. Both ofthe above d. Inflammatory carcinoma Dimpling of the skin, nippleretraction, or any other skin changes except those in T4b may occur inT1, T2 or T3 without affecting the classification. N REGIONAL LYMPHNODES NX Regional lymph nodes cannot be assessed (e.g., previouslyremoved) N0 No regional lymph node metastases N1 Metastasis to movableipsilateral axillary node(s) N2 Metastases to ipsilateral axillary nodesfixed to one another or to other structures N3 Metastases to ipsilateralinternal mammary lymph node(s) M DISTANT METASTASIS M0 No evidence ofdistant metastasis M1 Distant metastases (including metastases toipsilateral supraclavicular lymph nodes)

Any immunoassay, such as those described in Section 5.4.2 can be used tomeasure the amount of SGA-56M or SGA-56Mv polypeptide or soluble SGA-56Mor SGA-56Mv polypeptide as compared to a baseline level. This baselinelevel can be the amount that is established to be normally present inthe tissue or body fluid of subjects with various degrees of the diseaseor disorder. An amount present in the tissue or body fluid of thesubject that is similar to a standard amount, established to be normallypresent in the tissue or body fluid of the subject during a specificstage of cancer or breast cancer, is indicative of the stage of thedisease in the subject. The baseline level could also be the levelpresent in the subject prior to the onset of disease or the amountpresent during remission of the disease.

In specific embodiments of this aspect of the invention, measurements oflevels of the SGA-56M or SGA-56Mv polypeptide or soluble SGA-56M orSGA-56Mv polypeptide can be used in the detection of infiltrative ductalcarcinoma (IDC), the presence of metastases, or both. Increased levelsof SGA-56M or SGA-56Mv polypeptides or soluble SGA-56M or SGA-56Mvpolypeptide are associated with metastases.

In another embodiment of the invention, the measurement of solubleSGA-56M or SGA-56Mv polypeptide, intra-cellular SGA-56M or SGA-56Mvpolypeptide, fragments thereof or immunologically related molecules canbe used to differentially diagnose in a subject a particular diseasephenotype or physiological condition as distinct as from among two ormore phenotypes or physiological conditions. For example, measurementsof SGA-56M or SGA-56Mv polypeptide or soluble SGA-56M or SGA-56Mvpolypeptide levels may be used in the differential diagnosis ofinfiltrative ductal carcinoma, as distinguished from ductal carcinoma insitu or benign fibroadenomas. To this end, for example, the measuredamount of the SGA-56M or SGA-56Mv polypeptide is compared with theamount of the molecule normally present in the tissue, cells or bodyfluid of a subject with one of the suspected physiological conditions. Ameasured amount of the SGA-56M or SGA-56Mv polypeptide similar to theamount normally present in a subject with one of the physiologicalconditions, and not normally present in a subject with one or more ofthe other physiological conditions, is indicative of the physiologicalcondition of the subject.

As an alternative to measuring levels of SGA-56M or SGA-56Mvpolypeptides in the foregoing staging methods, levels of SGA-56M orSGA-56Mv transcript can be measured, for example by the methodsdescribed in Section 5.4.1, supra.

5.4.4. Monitoring the Effect of a Therapeutic Treatment

The present invention provides a method for monitoring the effect of atherapeutic treatment on a subject who has undergone the therapeutictreatment.

Clinicians very much need a procedure that can be used to monitor theefficacy of cancer treatments. SGA-56M or SGA-56Mv-encoded polypeptidesand/or transcripts can be identified and detected in breast cancerpatients or other cancer patients with different manifestations ofdisease, providing a sensitive assay to monitor therapy. The therapeutictreatments which may be evaluated according to the present inventioninclude but are not limited to radiotherapy, surgery, chemotherapy,vaccine administration, endocrine therapy, immunotherapy, and genetherapy, etc. The chemotherapeutic regimens include, but are not limitedto administration of drugs such as, for example, methotrexate,fluorouracil, cyclophosphamide, doxorubicin, and taxol. The endocrinetherapeutic regimens include, but are not limited to administration oftamoxifen, progestins, etc.

The method of the invention comprises measuring at suitable timeintervals before, during, or after therapy, the amount of an SGA-56M orSGA-56Mv transcript or polypeptide (including soluble polypeptide), orany combination of the foregoing. Any change or absence of change in theabsolute or relative amounts of the SGA-56M or SGA-56Mv gene productscan be identified and correlated with the effect of the treatment on thesubject.

In particular, the serum- or cell-associated levels of an SGA-56M orSGA-56Mv-encoded polypeptide relates to the severity of a cancer, suchas breast cancer, risk of metastasis of said cancer, and poor prognosis.Since serum- or cell-associated SGA-56M or SGA-56Mv polypeptide levelsare generally undetectable or negligible in normal individuals,generally, a decrease in the level of detectable SGA-56M or SGA-56Mvpolypeptide after a therapeutic treatment is associated with efficacioustreatment.

In a preferred aspect, the approach that can be taken is to determinethe levels of soluble or cell associated SGA-56M or SGA-56Mvpolyepeptide levels at different time points and to compare these valueswith a baseline level. The baseline level can be either the level of theSGA-56M or SGA-56Mv polypeptide present in normal, disease freeindividuals; and/or the levels present prior to treatment, or duringremission of disease, or during periods of stability. These levels canthen be correlated with the disease course or treatment outcome.

5.4.5. Prognostic Assays

The methods described herein can furthermore be utilized as prognosticassays to identify subjects having or at risk of developing cancer oranother disease or disorder associated with aberrant expression oractivity of an SGA-56M or SGA-56Mv polypeptide. For example, the assaysdescribed herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing cancer, e.g., breast cancer, or another disorderassociated with aberrant expression or activity of an SGA-56M orSGA-56Mv polypeptide. Thus, the present invention provides a method inwhich a test sample is obtained from a subject and an SGA-56M orSGA-56Mv polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of theinvention is detected, wherein the presence of the polypeptide ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant expression or activity ofthe SGA-56M or SGA-56Mv polypeptide, e.g., cancer. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue.

The prognostic assays described herein, for example, can be used toidentify a subject having or at risk of developing disorders such ascancers, for example, hormone-sensitive cancer such as breast cancer.

In another example, prognostic assays described herein can be used toidentify a subject having or at risk of developing related disordersassociated with expression of polypeptides or nucleic acids of theinvention.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat cancer or anotherdisease or disorder associated with aberrant expression or activity ofan SGA-56M or SGA-56Mv polypeptide. For example, such methods can beused to determine whether a subject can be effectively treated with aspecific agent or class of agents (e.g., agents of a type which decreaseactivity or expression level of an SGA-56M or SGA-56Mv transcript orpolypeptide). Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant expression or activity of theSGA-56M or SGA-56Mv transcript or polypeptide in which a test sample isobtained and the polypeptide or nucleic acid encoding the polypeptide isdetected (e.g., wherein the presence of the polypeptide or nucleic acidis diagnostic for a subject that can be administered the agent to treata disorder associated with aberrant expression or activity of theSGA-56M or SGA-56Mv transcript or polypeptide).

The methods of the invention can also be used to detect genetic lesionsor mutations in an SGA-56M or SGA-56Mv gene, thereby determining if asubject with the lesioned gene is at increased or reduced risk for adisorder characterized by aberrant expression or activity of apolypeptide of the invention, e.g., cancer. In one embodiment, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic lesion or mutation characterized by atleast one of an alteration affecting the integrity of a gene encoding anSGA-56M or SGA-56Mv polypeptide, or the mis-expression of the geneencoding an SGA-56M or SGA-56Mv polypeptide. For example, such geneticlesions or mutations can be detected by ascertaining the existence of atleast one of: 1) a deletion of one or more nucleotides from an SGA-56Mor SGA-56Mv gene; 2) an addition of one or more nucleotides to anSGA-56M or SGA-56Mv gene; 3) a substitution of one or more nucleotidesof an SGA-56M or SGA-56Mv gene i.e. a point mutation; 4) a chromosomalrearrangement of an SGA-56M or SGA-56Mv gene; 5) an alteration in thelevel of a messenger RNA transcript of an SGA-56M or SGA-56Mv gene; 6)an aberrant modification of an SGA-56M or SGA-56Mv gene, such as of themethylation pattern of the genomic DNA; 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of an SGA-56M orSGA-56Mv gene; 8) a non-wild type level of the protein encoded by anSGA-56M or SGA-56Mv gene; 9) an allelic loss of an SGA-56M or SGA-56Mvgene; and 10) an inappropriate post-translational modification of aprotein encoded by an SGA-56M or SGA-56Mv gene. As described herein,there are a large number of assay techniques known in the art that canbe used for detecting lesions in a gene.

In certain embodiments, methods for the detection of the lesion involvethe use of a probe/primer in a polymerase chain reaction (PCR) (See,e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR orRACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al. (1988) Science 241:1077; and Nakazawa et al.(1994) Proc Natl Acad Sci. USA 91:360), the latter of which can beparticularly useful for detecting point mutations in a gene (see, e.g.,Abravaya et al. (1995) Nucleic Acids Res. 23:675). These methods areuseful in the diagnosis and prognosis of cancer in a subject. Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to the selected gene underconditions such that hybridization and amplification of the gene or geneproduct (if present) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Mutations in a selected gene from a sample cell or tissue can also beidentified by alterations in restriction enzyme cleavage patterns. Forexample, sample and control DNA is isolated, amplified (optionally),digested with one or more restriction endonucleases, and fragment lengthsizes are determined by gel electrophoresis and compared. Differences infragment length sizes between sample and control DNA indicates mutationsin the sample DNA. Moreover, the use of sequence specific ribozymes(see, e.g., U.S. Pat. No. 5,498,531) can be used to score for thepresence of specific mutations by development or loss of a ribozymecleavage site.

In other embodiments, methods are provided whereby genetic mutations canbe identified by hybridizing a sample and control nucleic acids, e.g.,DNA or RNA, to high density arrays comprising hundreds or thousands ofoligonucleotides probes (Cronin et al. 1996, Human Mutation 7:244; Kozalet al. 1996, Nature Medicine 2:753). For example, genetic mutations canbe identified in two-dimensional arrays containing light-generated DNAprobes as described in Cronin et al., supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

Sequencing reactions known in the art can be used to directly sequencethe selected gene and detect mutations in the SGA-56M or SGA-56Mv geneby comparing the sequence of the sample nucleic acids with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert (Maxim and Gilbert, 1977, Proc Natl Acad. Sci. USA 74:560) orSanger (Sanger et al. 1977, Proc Natl Acad. Sci. USA 74:5463). Suchmethods are useful in the diagnosis and prognosis of a subject withcancer. It is also contemplated that any of a variety of automatedsequencing procedures can be utilized when performing the diagnosticassays (Naeve et al., 1995, BioTechniques 19:448), including sequencingby mass spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohenet al. 1996, Adv. Chromatogr. 36: 127; and Griffin et al., 1993, Appl.Biochem. Biotechnol. 38:147).

Furthermore, the presence of an SGA-56M or SGA-56Mv nucleic acidmolecule or polypeptide of the invention can be correlated with thepresence or expression level of other cancer-related proteins, such asfor example, an androgen receptor, estrogen receptor, adhesion molecules(e.g., E-cadherin), proliferation markers (e.g., MIB-1),tumor-suppressor genes (e.g., TP53, retinoblastoma gene product),vascular endothelial growth factor (Lissoni et al., 2000, Int J BiolMarkers. 15(4):308), Rad51 (Maacke et al., 2000, Int J Cancer.88(6):907), cyclin D1, BRCA1, BRCA2, or carcinoembryonic antigen.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one nucleic acid probeor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a gene encoding apolypeptide of the invention. Furthermore, any cell type or tissue,e.g., preferably cancerous breast cells or tissue, in which the SGA-56Mor SGA-56Mv gene is expressed, may be utilized in the prognostic assaysdescribed herein.

5.5. Screening for Modulators of SGA-56M and/or SGA-56Mv Activity

The present invention further provides methods for the identification ofcompounds that may, through their interaction with the SGA-56M and/orSGA-56Mv gene or SGA-56M and/or SGA-56Mv gene product, affect the onset,progression and metastatic spread of breast cancer and/or other cancers.

The following assays are designed to identify: (i) compounds that bindto SGA-56M and/or SGA-56Mv gene products; (ii) compounds that bind toother proteins that interact with an SGA-56M and/or SGA-56Mv geneproduct; (iii) compounds that interfere with the interaction of theSGA-56M and/or SGA-56Mv gene product with other proteins; and (iv)compounds that modulate the activity of an SGA-56M and/or SGA-56Mv gene(i.e., modulate the level of SGA-56M and/or SGA-56Mv gene expression,including transcription of the SGA-56M and/or SGA-56Mv gene and/ortranslation of its encoded transcript, and/or modulate SGA-56M and/orSGA-56Mv-encoded polypeptide activity).

Assays may additionally be utilized which identify compounds that bindto SGA-56M and/or SGA-56Mv gene regulatory sequences (e.g., promotersequences), which may modulate the level of SGA-56M and/or SGA-56Mv geneexpression (see e.g., Platt, K. A., 1994, J. Biol. Chem. 269:28558).

Such proteins that interact with SGA-56M and/or SGA-56Mv may be involvedin the onset, development and metastatic spread of breast cancer orother cancers. Accordingly, methods to modulate the expression leveland/or activity of a protein that interacts with SGA-56M and/or SGA-56Mvmay also present an effective approach toward modulating the expressionand/or activity of SGA-56M and/or SGA-56Mv.

The present invention also provides methods of using isolated SGA-56Mand/or SGA-56Mv nucleic acid molecules, or derivatives thereof, asprobes that can be used to screen for DNA-binding proteins, includingbut not limited to proteins that affect DNA conformation or modulatetranscriptional activity (e.g., enhancers, transcription factors). Inanother embodiment, such probes can be used to screen for RNA-bindingfactors, including but not limited to proteins, steroid hormones, orother small molecules. In yet another embodiment, such probes can beused to detect and identify molecules that bind or affect thepharmacokinetics or activity (e.g., enzymatic activity) of the SGA-56Mand/or SGA-56Mv gene or gene product. The protein- or nucleicacid-binding factors or transcriptional modulators identified by ascreening assay would provide an appropriate reagent for anti-cancertherapeutics.

In one embodiment, a screening assay of the invention can identify atest compound that is useful for increasing or decreasing thetranslation of one or both SGA-56M or SGA-56Mv ORFs, for example, bybinding to one or more regulatory elements in the 5′ untranslatedregion, the 3′ untranslated region, or the coding regions of the mRNA.Compounds that bind to mRNA can, inter alia, increase or decrease therate of mRNA processing, alter its transport through the cell, preventor enhance binding of the mRNA to ribosomes, suppressor proteins orenhancer proteins, or alter mRNA stability. Compounds that increase ordecrease mRNA translation, for example, can be used to treat or preventdisease. For example, diseases such as cancer, associated withoverproduction of proteins, such as SGA-56M and/or SGA-56Mv, can betreated and/or prevented by decreasing translation of the mRNA thatcodes for the protein, thus inhibiting production of the protein.

Accordingly, in one embodiment, a compound identified by a screeningassay of the invention inhibits the production of an SGA-56M and/orSGA-56Mv protein. In a further embodiment, the compound inhibits thetranslation of an SGA-56M and/or SGA-56Mv mRNA. In yet anotherembodiment, the compound inhibits transcription of the SGA-56M and/orSGA-56Mv gene.

The invention provides a method for identifying modulators, i.e.,candidate or test compounds or agents (e.g., peptides, peptidomimetics,small molecules or other drugs) which bind to the SGA-56M and/orSGA-56Mv gene product or fragments thereof or have a stimulatory orinhibitory effect on, for example, expression or activity of the SGA-56Mand/or SGA-56Mv gene product or fragments thereof.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of theSGA-56M and/or SGA-56Mv gene product, and for ameliorating symptoms ofbreast cancer or other types of cancer. Assays for testing theeffectiveness of compounds, identified by, for example, techniques suchas those described in Section 5.5.1, are discussed, below, in Section5.5.3. It is to be noted that the compositions of the invention includepharmaceutical compositions comprising one or more of the compoundsidentified via such methods. Such pharmaceutical compositions can beformulated, for example, as discussed, below, in Section 5.7.

5.5.1. In vitro Screening Assays for Compounds that Bind to The SGA-56Mand/or SGA-56Mv Gene Product

In vitro systems may be designed to identify compounds capable ofinteracting with, e.g., binding to, an SGA-56M and/or SGA-56Mv geneproduct of the invention. Compounds identified may be useful, forexample, in modulating the activity of wild type and/or mutant SGA-56Mand/or SGA-56Mv gene products, may be useful in elaborating thebiological function of the SGA-56M and/or SGA-56Mv gene product, may beutilized in screens for identifying compounds that disrupt normalSGA-56M and/or SGA-56Mv gene product interactions, or may in themselvesdisrupt such interactions. Thus said compounds would be useful intreating, preventing and/or diagnosing cancer. In a particularembodiment said compounds are useful in the treatment, prevention anddiagnosis of breast cancer.

The principle of the assays used to identify compounds that interactwith the SGA-56M and/or SGA-56Mv gene product involves preparing areaction mixture of the SGA-56M and/or SGA-56Mv gene product and thetest compound under conditions and for a time sufficient to allow thetwo components to interact with, e.g., bind to, thus forming a complex,which can represent a transient complex, which can be removed and/ordetected in the reaction mixture. These assays can be conducted in avariety of ways. For example, one method to conduct such an assay wouldinvolve anchoring SGA-56M or SGA-56Mv gene product or the test substanceonto a solid phase and detecting SGA-56M or SGA-56Mv gene product/testcompound complexes anchored on the solid phase at the end of thereaction. In one embodiment of such a method, the SGA-56M or SGA-56Mvgene product may be anchored onto a solid surface, and the testcompound, which is not anchored, may be labeled, either directly orindirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific or selective for the protein to be immobilized may beused to anchor the protein to the solid surface. The latter methodprovides for presentation of the protein in a known orientation. Thesurfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for an SGA-56Mand/or SGA-56Mv gene product or the test compound to anchor anycomplexes formed in solution, and a labeled antibody specific for theother component of the possible complex to detect anchored complexes.

5.5.2 Assays for Proteins that Interact with an SGA-56M or SGA-56Mv GeneProduct

Any method suitable for detecting protein-protein interactions may beemployed for identifying SGA-56M and/or SGA-56Mv protein-proteininteractions. Proteins that interact with SGA-56M and/or SGA-56Mv willbe potential therapeutics for the treatment of cancer. Thus the assaysdescribed below are useful in identifying proteins that can be used inmethods to treat cancer. Proteins that interact with SGA-56M and/orSGA-56Mv can also be used in the diagnosis of cancer. Thus, the assaysdescribed below are also useful in methods to diagnose cancer.

Among the traditional methods that may be employed areco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns (e.g., size exclusionchromatography). Utilizing procedures such as these allows for theisolation of intracellular proteins that interact with SGA-56M and/orSGA-56Mv gene products. Once isolated, such an intracellular protein canbe identified and can, in turn, be used, in conjunction with standardtechniques, to identify additional proteins with which it interacts. Forexample, at least a portion of the amino acid sequence of anintracellular protein or a protein having an intracellular domain whichinteracts with the SGA-56M and/or SGA-56Mv gene product can beascertained using techniques well known to those of skill in the art,such as via the Edman degradation technique (see, e.g., Creighton, 1983,Proteins: Structures and Molecular Principles, W.H. Freeman & Co., NewYork, pp. 34-49). The amino acid sequence obtained may be used as aguide for the generation of oligonucleotide mixtures that can be used toscreen for gene sequences encoding such proteins. Screening may beaccomplished, for example, by standard hybridization or PCR techniques.Techniques for the generation of oligonucleotide mixtures and screeningare well known. (See, e.g., Ausubel, supra, and PCR Protocols: A Guideto Methods and Applications, 1990, Innis, M. et al., eds. AcademicPress, Inc., New York).

Additionally, methods may be employed which result in the simultaneousidentification of genes which encode a protein interacting with theSGA-56M and/or SGA-56Mv protein. These methods include, for example,probing expression libraries with labeled SGA-56M or SGA-56Mv protein ina manner similar to the well known technique of antibody probing ofλgt11 libraries.

One method that detects protein interactions in vivo, the two-hybridsystem, may also be used. Many versions of this system have beendescribed (see, e.g., Chien et al., 1991, supra) and some arecommercially available from Clontech (Palo Alto, Calif.).

5.5.3. Assays for Compounds that Interfere with SGA-56M and/or SGA-56MvInteraction

The SGA-56M and/or SGA-56Mv gene product may, in vivo, interact with oneor more macromolecules, such as proteins or nucleic acids. Suchmacromolecules are referred to herein as “interacting partners” or“specific binding partners”. Compounds that disrupt an association ofSGA-56M and/or SGA-56Mv with interacting partner(s) may be useful inregulating the activity of the SGA-56M and/or SGA-56Mv gene product,including mutant SGA-56M and/or SGA-56Mv gene products. Such compoundsmay include, but are not limited to molecules such as peptides, and thelike, as described, for example, in Section 5.5.1., which would becapable of interacting with SGA-56M and/or SGA-56Mv polypeptides. Thusthe assays described below are useful for identifying proteins and/ornucleic acids that can be used in methods to treat cancer. Proteins andnucleic acids that interact with SGA-56M and/or SGA-56Mv can also beused in the diagnosis of cancer, e.g., breast cancer. Thus the assaysdescribed below are also useful in methods to diagnose cancer, e.g.,breast cancer.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the SGA-56M and/or SGA-56Mv geneproduct and an interacting partner or partners involves preparing areaction mixture containing the SGA-56M and/or SGA-56Mv gene product,and the interacting partner under conditions and for a time sufficientto allow the two to interact and bind, thus forming a complex. In orderto test a compound for inhibitory activity, the reaction mixture isprepared in the presence and absence of the test compound. The testcompound may initially be included in the reaction mixture, or may beadded at a time subsequent to the addition of SGA-56M and/or SGA-56Mvgene product and an interacting partner. Control reaction mixtures areincubated without the test compound or with a control compound. Theformation of any complexes between an SGA-56M and/or SGA-56Mv geneprotein and an interacting partner is then detected. The formation of acomplex in the control reaction, but not in a reaction mixturecomprising a test compound, indicates that the compound interferes withthe interaction of the SGA-56M and/or SGA-56Mv gene product and theinteracting partner. Additionally, complex formation within reactionmixtures containing the test compound and normal SGA-56M and/or SGA-56Mvgene protein may also be compared to complex formation within reactionmixtures containing the test compound and a mutant form of either anSGA-56M and/or SGA-56Mv geneproduct. This comparison may be important inthose cases wherein it is desirable to identify compounds that disruptinteractions of mutant but not normal SGA-56M and/or SGA-56Mv geneproteins.

The assay for compounds that interfere with the interaction of theSGA-56M and/or SGA-56Mv gene products and interacting partners can beconducted in a heterogeneous or homogeneous format. Heterogeneous assaysinvolve anchoring either an SGA-56M or SGA-56Mv gene product or thebinding partner onto a solid phase and detecting complexes anchored onthe solid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the SGA-56M orSGA-56Mv gene products and the interacting partners, e.g., bycompetition, can be identified by conducting the reaction in thepresence of the test substance; i.e., by adding the test substance tothe reaction mixture prior to or simultaneously with the SGA-56M orSGA-56Mv gene protein and intracellular interacting partner.Alternatively, test compounds that disrupt preformed complexes, e.g.,compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the SGA-56M or SGA-56Mv geneproduct or the interacting partner, is anchored onto a solid surface,while the non-anchored species is labeled, either directly orindirectly. In practice, microtiter plates are conveniently utilized.The anchored species may be immobilized by non-covalent or covalentattachments. Non-covalent attachment may be accomplished simply bycoating the solid surface with a solution of the SGA-56M or SGA-56Mvgene product or interacting partner and drying. Alternatively, animmobilized antibody, for example, specific for the species to beanchored may be used to anchor the species to the solid surface. Thesurfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, may bedirectly labeled or indirectly labeled with, for example, a labeledanti-Ig antibody). Depending upon the order of addition of reactioncomponents, test compounds which inhibit complex formation or whichdisrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected. Thereaction, for example, may be executed using an immobilized antibodyspecific for one of the interacting components to anchor any complexesformed in solution, and the labeled antibody specific for the otherpartner to detect anchored complexes. Again, depending upon the order ofaddition of reactants to the liquid phase, test compounds which inhibitcomplex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the SGA-56M or SGA-56Mvgene protein and the interacting partner is prepared in which either theSGA-56M or SGA-56Mv gene product or its interacting partner is labeled,but the signal generated by the label is quenched due to complexformation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein). Theaddition of a test substance that competes with and displaces one of thespecies from the preformed complex will result in the generation of asignal above background. In this way, test substances that disruptSGA-56M and/or SGA-56Mv gene product association with an interactingpartner can be identified.

In a particular embodiment, the SGA-56M or SGA-56Mv gene product can beprepared for immobilization using recombinant DNA techniques describedin Section 5.1, above. For example, the SGA-56M or SGA-56Mv codingregion can be fused to a glutathione-5-transferase (GST) gene using afusion vector, such as pGEX-5×-1, in such a manner that its interactingactivity is maintained in the resulting fusion protein. The interactingpartner can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art and described above, in Section5.2. This antibody can be labeled with the radioactive isotope ¹²⁵I, forexample, by methods routinely practiced in the art. In a heterogeneousassay, e.g., the GST-SGA-56M or GST-SGA-56Mv fusion protein can beanchored to glutathione-agarose beads. The interacting partner can thenbe added in the presence or absence of the test compound in a mannerthat allows interaction, e.g., binding, to occur. At the end of thereaction period, unbound material can be washed away, and the labeledmonoclonal antibody can be added to the system and allowed to bind tothe complexed components. The interaction between the SGA-56M orSGA-56Mv gene protein and the interacting partner can be detected bymeasuring the amount of radioactivity that remains associated with theglutathione-agarose beads. Alternative means may be applied for suchapproaches, including the generation of detectable and distinguishablefusion proteins comprising each of the interacting proteins, e.g.,SGA-56M-GST and a His tagged version of an SGA-56M interacting protein.A successful inhibition of the interaction by the test compound willresult in a decrease in measured radioactivity.

Alternatively, the GST-SGA-56M or GST-SGA-56Mv fusion protein and theintracellular interacting partner can be mixed together in liquid in theabsence of the solid glutathione-agarose beads. The test compound can beadded either during or after the species are allowed to interact. Thismixture can then be added to the glutathione-agarose beads and unboundmaterial is washed away. The extent of inhibition of SGA-56M or SGA-56Mvgene product/binding partner interaction can be detected by addition ofa labeled antibody, for example, and measuring the radioactivityassociated with the beads.

It will be appreciated that the above assays may be preformed with amixture of SGA-56M and SGA-56Mv gene product. The ratio at which thedifferent gene products are mixed may be varied according to theapplication.

5.5.4. Cell-Based Assays for SGA-56M or SGA-56Mv Activity

Cell-based methods are presented herein which identify compounds capableof treating breast cancer and other cancers by modulating SGA-56M and/orSGA-56Mv activity or expression levels. Specifically, such assaysidentify compounds that affect SGA-56M and/or SGA-56Mv dependentprocesses, such as, but not limited to changes in cell morphology, celldivision, differentiation, adhesion, motility, phosphorylation, ordephosphorylation of cellular proteins. Such assays can also be used toidentify compounds that affect SGA-56M and/or SGA-56Mv expression levelsor gene activity directly. Compounds identified via such methods can,for example, be utilized in methods for treating breast cancer and othercancers and metastasis thereof.

In one embodiment, an assay is a cell-based assay in which a cell thatexpresses a membrane-bound form of the SGA-56M or SGA-56Mv gene product,or a biologically active portion thereof, on the cell surface iscontacted with a test compound and the ability of the test compound tobind to the polypeptide determined. In another embodiment the SGA-56M orSGA-56Mv gene product is cytosolic. The cell, for example, can be ayeast cell or a cell of mammalian origin. Determining the ability of thetest compound to bind to the polypeptide can be accomplished, forexample, by coupling the test compound with a radioisotope or enzymaticlabel such that binding of the test compound to the polypeptide orbiologically active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radio-emission or byscintillation counting. Alternatively, test compounds can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product. In apreferred embodiment, the assay comprises contacting a cell whichexpresses a membrane-bound form of a polypeptide of the invention, or abiologically active portion thereof, on the cell surface with a knowncompound which binds the polypeptide to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the polypeptide, whereindetermining the ability of the test compound to interact with thepolypeptide comprises determining the ability of the test compound topreferentially bind to the polypeptide or a biologically active portionthereof as compared to a reference compound.

In another embodiment, the cell-based assays are based on expression ofthe SGA-56M or SGA-56Mv gene product in a mammalian cell and measuringthe SGA-56M and/or SGA-56Mv-dependent process. Any mammalian cells thatcan express the SGA-56M and/or SGA-56Mv gene and allow the functioningof the SGA-56M and/or SGA-56Mv gene product can be used, in particular,cancer cells derived from the breast, such as MCF-7, BT483, Hs578T,HTB26, BT20 and T47D. Normal mammary gland cell lines such as, forexample, CRL7030 and Hs578Bst, may also be used provided that an SGA-56Mand/or SGA-56Mv gene product is produced. Other mammalian cell linesthat can be used include, but are not limited to, CHO, HeLa, NIH3T3, andVero cells. Recombinant expression of the SGA-56M and/or SGA-56Mv genein these cells can be achieved by methods described in Section 5.2. Inthese assays, cells producing functional SGA-56M and/or SGA-56Mv geneproducts are exposed to a test compound for an interval sufficient forthe compound to modulate the activity of the SGA-56M and/or SGA-56Mvgene product. The activity of SGA-56M and/or SGA-56Mv gene product canbe measured directly or indirectly through the detection or measurementof SGA-56M and/or SGA-56Mv-dependent cellular processes. As a control, acell not producing the SGA-56M and/or SGA-56Mv gene product may be usedfor comparisons. Depending on the cellular process, any techniques knownin the art may be applied to detect or measure it

In another embodiment a cell or cell line that is capable of expressingSGA-56M and/or SGA-56Mv is contacted with a test compound that isbelieved to modulate expression of the SGA-56M and/or SGA-56Mv gene.Expression levels of the SGA-56M and/or SGA-56Mv gene can be monitoredin the presence or absence of the test compound. Alternatively,expression levels can be monitored in the presence of a test compound ascompared to expression levels of the SGA-56M and/or SGA-56Mv gene in thepresence of a control compound or a placebo. Any method known in the artcan be used to monitor SGA-56M and/or SGA-56Mv gene expression. As anexample, but not as a limitation, such methods can include analysis byWestern blot, Northern blot, and real-time quantitative RT-PCR.

In yet another embodiment, cells which express the SGA-56M and/orSGA-56Mv gene product, e.g., MCF-7 cells are made permeable, e.g., bytreatment with a mild detergent and exposed to a test compound. Bindingof the test compound can be detected directly (e.g., radioactivelylabeling the test compound) or indirectly (e.g., antibody detection) orby any means known in the art.

Any compound can be used in a cell-based assay to test if it affectsSGA-56M and/or SGA-56Mv activity or expression levels. The compound canbe a protein, a peptide, a nucleic acid, an antibody or fragmentthereof, a small molecule, an organic molecule or an inorganic molecule.(e.g., steroid, pharmaceutical drug). A small molecule is considered anon-peptide compound with a molecular weight of less than 500 daltons.

5.6. Methods for Treatment of Cancer

Described below are methods and compositions for treating cancer, e.g.,breast or lung cancer, using the SGA-56M and/or SGA-56Mv gene or geneproduct as a therapeutic target. The outcome of a treatment is to atleast produce in a treated subject a healthful benefit, which in thecase of cancer, including breast cancer, includes but is not limited toremission of the cancer, palliation of the symptoms of the cancer,and/or control of metastatic spread of the cancer.

All such methods comprise methods for modulating SGA-56M and/or SGA-56Mvgene activity and/or expression which in turn modulate the phenotype ofthe treated cell and tumorigenic potential.

As discussed, above, successful treatment of breast cancer or othercancers can be effected through techniques that serve to decreaseSGA-56M and/or SGA-56Mv activity. Activity can be decreased, forexample, bydirectly decreasing SGA-56M and/or SGA-56Mv gene productactivity and/or by decreasing the level of SGA-56M and/or SGA-56Mv geneexpression. Thus the invention provides methods of treating a subjectwith cancer by administering to said subject a therapeutically effectiveamount of a compound that antagonizes an SGA-56M and/or SGA-56Mv geneproduct

For example, compounds such as those identified through assaysdescribed, above, in Section 5.5, above, which decrease SGA-56M and/orSGA-56Mv activity can be used in accordance with the invention to treatbreast cancer or other cancers. As discussed in Section 5.5, above, suchmolecules can include, but are not limited to proteins, nucleic acids,peptides, including soluble peptides, and small organic or inorganicmolecules, and can be referred to as SGA-56M and/or SGA-56Mvantagonists. Techniques for the determination of effective doses andadministration of such compounds are described, below, in Section 5.7.

Further, antisense and ribozyme molecules which inhibit SGA-56M and/orSGA-56Mv gene expression can also be used in accordance with theinvention to reduce the level of SGA-56M and/or SGA-56Mv geneexpression, thus effectively reducing the level of SGA-56M and/orSGA-56Mv gene product present, thereby decreasing the level of SGA-56Mand/or SGA-56Mv activity. The invention therefore relates to apharmaceutical composition comprising an antisense or ribozyme moleculewith specificity for an SGA-56M and/or SGA-56Mv gene product. Stillfurther, triple helix molecules can be utilized in reducing the level ofSGA-56M and/or SGA-56Mv gene activity. Such molecules can be designed toreduce or inhibit either wild type, or if appropriate, mutant targetgene activity. Small organic or inorganic molecules can also be used toinhibit SGA-56M and/or SGA-56Mv gene expression and/or inhibitproduction and/or activity of an SGA-56M and/or SGA-56Mv gene product.Techniques for the production and use of such molecules are well knownto those of skill in the art.

5.6.1. Antisense Molecules

Anti-sense nucleic acid molecules which are complementary to nucleicacid sequences contained within the SGA-56M or SGA-56Mv gene as shown inFIG. 1 (SEQ ID NO: 1) and FIG. 2 (SEQ ID NO: 3), including but notlimited to anti-sense nucleic acid molecules complementary to (SEQ IDNO: 1) and (SEQ ID NO: 3), can be used to treat any cancer, in which theexpression level of the SGA-56M or SGA-56Mv gene is elevated incancerous cells or tissue as compared to normal cells or tissue or apredetermined non-cancerous standard. Thus in one embodiment of theinvention a method of treating breast cancer is provided whereby apatient suffering from breast cancer is treated with a therapeuticallyeffective amount of an SGA-56M or SGA-56Mv anti-sense nucleic acidmolecule.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to SGA-56M or SGA-56Mv gene mRNA. Theantisense oligonucleotides win bind to the complementary SGA-56M orSGA-56Mv gene mRNA transcripts and prevent translation. Absolutecomplementarity, although preferred, is not required. A sequence“complementary” to a portion of an RNA, as referred to herein, means asequence having sufficient complementarity to be able to hybridize withthe non-poly A portion of the RNA, forming a stable duplex; in the caseof double-stranded antisense nucleic acids, a single strand of theduplex DNA may thus be tested, or triplex formation may be assayed. Theability to hybridize will depend on both the degree of complementarityand the length of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with regard to anucleic acid target it may contain and still form a stable duplex (ortriplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have also been shown to be effective at inhibitingtranslation of mRNAs as well. (See generally, Wagner, 1994, Nature372:333). Thus, oligonucleotides complementary to the 5′-non-translatedregion, the 3′-non-translated region, or the non-translated, non-codingregion between the SGA-56M or SGA-56Mv open reading frame of the SGA-56Mor SGA-56Mv gene (referred to herein after as the “intervening region”,as shown, for example, in FIG. 1, could be used in an antisense approachto inhibit translation of endogenous SGA-56M or SGA-56Mv gene mRNA.

Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould ideally include the complement of the AUG start codon. Antisenseoligonucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could be used in accordance with theinvention. Whether designed to hybridize to the 5′-, 3′-, intervening,or coding region of SGA-56M and/or SGA-56Mv gene mRNA, antisense nucleicacids should be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared to those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA86:6553; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648; PCTPublication No. WO88/09810, published Dec. 15, 1988) or the blood-brainbarrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25,1988), hybridization-triggered cleavage agents. (see, e.g., Krol et al.,1988, BioTechniques 6:958) or intercalating agents. (see, e.g., Zon,1988, Pharm. Res. 5:539). To this end, the oligonucleotide may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625). The oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett.215:327).

The SGA-56M antisense nucleic acid sequence can comprise the complementof any contiguous segment within the sequence of the SGA-56M gene (SEQID NO: 1).

In one embodiment of the present invention, the SGA-56M antisensenucleic acid sequence is about 50 bp in length. In certain specificembodiments, the SGA-56M antisense nucleic acid sequence comprises thesequence complementary to any contiguous block of 50, 100, 200, or 400nucleotides of SEQ ID NO: 1.

The SGA-56Mv antisense nucleic acid sequence can comprise the complementof any contiguous segment within the sequence of the SGA-56Mv gene (SEQID NO: 3).

In one embodiment of the present invention, the SGA-56Mv antisensenucleic acid sequence is about 50 bp in length. In certain specificembodiments, the SGA-56Mv antisense nucleic acid sequence comprises thesequence complementary to any contiguous block of 50, 100, 200, or 400nucleotides of SEQ ID NO: 3.

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. SciU.S.A. 85:7448), etc.

While antisense nucleotides complementary to the SGA-56M coding regioncould be used, those complementary to the transcribed untranslatedregion are most preferred.

The antisense molecules should be delivered to cells that express theSGA-56M and/or SGA-56Mv gene in vivo. A number of methods have beendeveloped for delivering antisense DNA or RNA to cells; e.g., antisensemolecules can be injected directly into the tissue site, or modifiedantisense molecules, designed to target the desired cells (e.g.,antisense linked to peptides or antibodies that specifically bindreceptors or antigens expressed on the target cell surface) can beadministered systemically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in a patient results in the transcription ofsufficient amounts of single stranded RNAs that will form complementarybase pairs with the endogenous SGA-56M and/or SGA-56Mv gene transcriptsand thereby prevent translation of the SGA-56M and/or SGA-56Mv genemRNA. For example, a vector can be introduced in vivo such that it canbe taken up by a cell and direct the transcription of an antisense RNA.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be a plasmid, viral vector, or otherconstruct known in the art, used for replication and expression inmammalian cells. Expression of the sequence encoding the antisense RNAcan be by any promoter known in the art to function in mammalian,preferably human cells. Such promoters can be inducible or constitutive.Such promoters include but are not limited to: the SV40 early promoterregion (Bernoist and Chambon, 1981, Nature 290:304), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., 1980, Cell 22:787), the herpes thymidine kinase promoter (Wagneret al., 1981, Proc. Natl. Acad. Sci. USA 78:1441), the regulatorysequences of the metallothionein gene (Brinster et al., 1982, Nature296:39), etc. Any type of plasmid, cosmid, YAC or viral vector can beused to prepare the recombinant DNA construct that can be introduceddirectly into the tissue site. Alternatively, viral vectors can be usedwhich selectively infect the desired tissue.

The effective dose of SGA-56M and/or SGA-56Mv antisense oligonucleotideto be administered during a treatment cycle ranges from about 0.01 to0.1, 0.1 to 1, or 1 to 10 mg/kg/day. The dose of SGA-56M and/or SGA-56Mvantisense oligonucleotide to be administered may depend on the mode ofadministration. For example, intravenous administration of an SGA-56Mand/or SGA-56Mv antisense oligonucleotide would likely result in asignificantly higher systemic dose than a full body dose resulting froma local implant comprising a pharmaceutical composition comprisingSGA-56M and/or SGA-56Mv antisense oligonucleotide. In one embodiment, anSGA-56M and/or SGA-56Mv antisense oligonucleotide is administeredsubcutaneously at a dose of 0.01 to 10 mg/kg/day. In another embodiment,an SGA-56M and/or SGA-56Mv antisense oligonucleotide is administeredintravenously at a dose of 0.01 to 10 mg/kg/day. In yet anotherembodiment, an SGA-56M and/or SGA-56Mv antisense oligonucleotide isadministered locally at a dose of 0.01 to 10 mg/kg/day. It will beevident to one skilled in the art that local administrations can resultin lower total body doses. For example, local administration methodssuch as intratumor administration, intraocular injection, orimplantation, can produce locally high concentrations of SGA-56M and/orSGA-56Mv antisense oligonucleotide, but represent a relatively low dosewith respect to total body weight. Thus, in such cases, localadministration of an SGA-56M and/or SGA-56Mv antisense oligonucleotideis contemplated to result in a total body dose of about 0.01 to 5mg/kg/day.

In another embodiment, a particularly high dose of SGA-56M and/orSGA-56Mv antisense oligonucleotide, which ranges from about 10 to 50mg/kg/day, is administered during a treatment cycle.

Moreover, the effective dose of a particular SGA-56M and/or SGA-56Mvantisense oligonucleotide may depend on additional factors, includingthe type of disease, the disease state or stage of disease,oligonucleotide toxicity, oligonucleotide stability, theoligonucleotides rate of uptake by cancer cells, as well as the weight,age, and health of the individual to whom the antisense oligonucleotideis to be administered. Because of the many factors present in vivo thatmay interfere with the action or biological activity of an SGA-56Mand/or SGA-56Mv antisense oligonucleotide, one of ordinary skill in theart can appreciate that an effective amount of an SGA-56M and/orSGA-56Mv antisense oligonucleotide may vary for each individual.

In another embodiment, an SGA-56M and/or SGA-56Mv antisenseoligonucleotide is at a dose which results in circulating plasmaconcentrations of an SGA-56M and/or SGA-56Mv antisense oligonucleotidewhich is at least 50 nM (nanomolar). As will be apparent to the skilledartisan, lower or higher plasma concentrations of an SGA-56M and/orSGA-56Mv antisense oligonucleotide may be preferred depending on themode of administration. For example, plasma concentrations of an SGA-56Mand/or SGA-56Mv antisense oligonucleotide of at least 50 nM can beappropriate in connection with intravenous, subcutaneous, intramuscular,controlled release, and oral administration methods, to name a few. Inanother example, relatively low circulating plasma levels of an SGA-56Mand/or SGA-56Mv antisense oligonucleotide can be desirable, however,when using local administration methods such as, for example, intratumoradministration, intraocular administration, or implantation, whichnevertheless can produce locally high, clinically effectiveconcentrations of SGA-56M and/or SGA-56Mv antisense oligonucleotide.

The high dose may be achieved by several administrations per cycle.Alternatively, the high dose may be administered in a single bolusadministration. A single administration of a high dose may result incirculating plasma levels of SGA-56M and/or SGA-56Mv antisenseoligonucleotide that are transiently much higher than 50 nM.

Additionally, the dose of an SGA-56M and/or SGA-56Mv antisenseoligonucleotide may vary according to the particular SGA-56M and/orSGA-56Mv antisense oligonucleotide used. The dose employed is likely toreflect a balancing of considerations, among which are stability,localization, cellular uptake, and toxicity of the particular SGA-56Mand/or SGA-56Mv antisense oligonucleotide. For example, a particularchemically modified SGA-56M and/or SGA-56Mv antisense oligonucleotidemay exhibit greater resistance to degradation, or may exhibit higheraffinity for the target nucleic acid, or may exhibit increased uptake bythe cell or cell nucleus, any of which properties may permit the use oflower doses. In yet another example, a particular chemically modifiedSGA-56M and/or SGA-56Mv antisense oligonucleotide may exhibit lowertoxicity than other antisense oligonucleotides, and therefore can beused at high doses. Thus, for a given SGA-56M and/or SGA-56Mv antisenseoligonucleotide, an appropriate dose to administer can be relativelyhigh or relatively low. The invention contemplates the continuedassessment of optimal treatment schedules for particular species ofSGA-56M and/or SGA-56Mv antisense oligonucleotides. The daily dose canbe administered in one or more treatments.

A “low dose” or “reduced dose” refers to a dose that is below thenormally administered range, i.e., below the standard dose as suggestedby the Physicians' Desk Reference. 54^(th) , Edition (2000) or a similarreference. Such a dose can be sufficient to inhibit cell proliferation,demonstrate ameliorative effects in a human, or demonstrate efficacywith fewer side effects as compared to standard cancer treatments.Normal dose ranges used for particular therapeutic agents and standardcancer treatments employed for specific diseases can be found in thePhysicians' Desk Reference, 54^(th) , Edition (2000) or in Cancer:Principles & Practice of Oncology, DeVita, Jr., Hellman, and Rosenberg(eds.) 2nd edition, Philadelphia, Pa.: J. B. Lippincott Co., 1985.

Reduced doses of an SGA-56M or SGA-56Mv nucleic acid molecule, SGA-56Mor SGA-56Mv polypeptide, SGA-56M and/or SGA-56Mv antagonist, and/orcombination therapeutic can demonstrate reduced toxicity, such thatfewer side effects and toxicities are observed in connection withadministering an SGA-56M and/or SGA-56Mv antagonist and one or morecancer therapeutics for shorter duration and/or at lower dosages whencompared to other treatment protocols and dosage formulations, includingthe standard treatment protocols and dosage formulations as described inthe Physicians' Desk Reference. 54^(th) Edition (2000) or in Cancer:Principles & Practice of Oncology, DeVita, Jr., Hellman, and Rosenberg(eds.) 2nd edition, Philadelphia, Pa.: J. B. Lippincott Co., 1985.

A “treatment cycle” or “cycle” refers to a period during which at leastone therapeutic or sequence of therapeutics is administered. In someinstances, one treatment cycle may be desired, such as, for example, inthe case where a significant therapeutic effect is obtained after onetreatment cycle. The present invention contemplates at least onetreatment cycle, generally preferably more than one treatment cycle.

Other factors to be considered in determining an effective dose of anSGA-56M and/or SGA-56Mv antisense oligonucleotide include whether theoligonucleotide will be administered in combination with othertherapeutics. In such cases, the relative toxicity of the othertherapeutics may indicate the use of an SGA-56M and/or SGA-56Mvantisense oligonucleotide at low doses. Alternatively, treatment with ahigh dose of SGA-56M and/or SGA-56Mv antisense oligonucleotide canresult in combination therapies with reduced doses of therapeutics. In aspecific embodiment, treatment with a particularly high dose of SGA-56Mand/or SGA-56Mv antisense oligonucleotide can result in combinationtherapies with greatly reduced doses of cancer therapeutics. Forexample, treatment of a patient with 10, 20, 30, 40, or 50 mg/kg/day ofan SGA-56M and/or SGA-56Mv antisense oligonucleotide can furtherincrease the sensitivity of a subject to cancer therapeutics. In suchcases, the particularly high dose of SGA-56M and/or SGA-56Mv antisenseoligonucleotide is combined with, for example, a greatly shortenedradiation therapy schedule. In another example, the particularly highdose of an SGA-56M and/or SGA-56Mv antisense oligonucleotide producessignificant enhancement of the potency of cancer therapeutic agents.

Additionally, the particularly high doses of SGA-56M and/or SGA-56Mvantisense oligonucleotide may further shorten the period ofadministration of a therapeutically effective amount of SGA-56M and/orSGA-56Mv antisense oligonucleotide and/or additional therapeutic, suchthat the length of a treatment cycle is much shorter than that of thestandard treatment.

The invention contemplates other treatment regimens depending on theparticular SGA-56M and/or SGA-56Mv antisense oligonucleotide to be used,or depending on the particular mode of administration, or depending onwhether an SGA-56M and/or SGA-56Mv antisense oligonucleotide isadministered as part of a combination therapy, e.g., in combination witha cancer therapeutic agent. The daily dose can be administered in one ormore treatments.

5.6.2. Ribozyme Molecules

Ribozyme molecules that are complementary to RNA sequences encoded forby the SGA-56M or SGA-56Mv gene as shown in FIG. 1 and FIG. 2 can beused to treat any cancer, including breast cancer. Ribozymes areenzymatic RNA molecules capable of catalyzing the specific cleavage ofRNA (for a review see, for example Rossi, J., 1994, Current Biology4:469). The mechanism of ribozyme action involves sequence specifichybridization of the ribozyme molecule to complementary target RNA,followed by endonucleolytic cleavage. Ribozyme molecules include one ormore sequences complementary to the target gene mRNA, and the well knowncatalytic sequence responsible for mRNA cleavage (See U.S. Pat. No.5,093,246). As such, within the scope of the invention are engineeredhammerhead motif ribozyme molecules that specifically and efficientlycatalyze endonucleolytic cleavage of RNA sequences encoding target geneproteins. Ribozyme molecules designed to catalytically cleave SGA-56M orSGA-56Mv mRNA transcripts can also be used to prevent translation ofSGA-56M or SGA-56Mv mRNA. (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222). While ribozymes that cleave mRNA at site-specific recognitionsequences can be used to destroy SGA-56M or SGA-56Mv mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, 1988, Nature 334:585.Preferably the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the SGA-56M or SGA-56Mv mRNA; Le., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Cech andcollaborators (Zaug et al., 1984, Science 224:574; Zaug and Cech, 1986,Science 231:470; Zaug et al., 1986, Nature 324:429; publishedInternational patent application No. WO 88/04300 by University PatentsInc.; Been and Cech, 1986, Cell 47:207). The Cech-type ribozymes have aneight base pair active site that hybridizes to a target RNA sequencewhereafter cleavage of the target RNA takes place. The inventionencompasses those Cech-type ribozymes that target eight base-pair activesite sequences that are present in an SGA-56M or SGA-56Mv gene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells that express the SGA-56M or SGA-56Mv genein vivo. A preferred method of delivery involves using a DNA construct“encoding” the ribozyme under the control of a strong constitutive polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous SGA-56M orSGA-56Mv transcripts and inhibit translation. Ribozymes, unlikeantisense molecules, are catalytic and require a lower intracellularconcentration to ensure effectiveness.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention can be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculescan be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well-known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

5.6.3. Therapeutic Antibodies

Antibodies exhibiting capability to downregulate SGA-56M and/or SGA-56Mvgene product activity can be utilized to treat breast cancer and othercancers wherein SGA-56M and/or SGA-56Mv expression levels are elevated.Antibodies immunologically specific for wild type or mutant SGA-56Mand/or SGA-56Mv proteins, or peptides corresponding to portions of theproteins can be generated using standard techniques described in Section5.3. Such antibodies include, but are not limited to polyclonal,monoclonal, Fab fragments, single chain antibodies, chimeric antibodies,and the like.

Antibodies that recognize any epitope on the SGA-56M and/or SGA-56Mvprotein can be used as therapeutics for the treatment and/or preventionof cancer.

Because SGA-56M and SGA-56Mv are generally expressed as intracellularproteins, it is preferred that internalizing antibodies be used.However, lipofectin or liposomes can be used to deliver an SGA-56Mand/or SGA-56Mv antibody or a fragment of an Fab region thereof intocells. Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to an SGA-56M and/or SGA-56Mv polypeptide ispreferred. For example, peptides having an amino acid sequencecorresponding to the domain of the variable region of an SGA-56M and/orSGA-56Mv specific antibody can be used. Such peptides can be synthesizedchemically or produced via recombinant DNA technology using methods wellknown in the art (e.g., see Creighton, 1983, supra; and Sambrook et al.,1989, supra). Alternatively, single chain antibodies, such asneutralizing antibodies, can also be administered. Such single chainantibodies can be administered, for example, by expressing nucleotidesequences encoding single-chain antibodies within the target cellpopulation by utilizing, for example, techniques such as those describedin Marasco et al. (Marasco, et al., 1993, Proc. Natl. Acad. Sci. USA90:7889).

The invention also contemplates methods wherein SGA-56M and/or SGA-56Mvantibodies conjugated to a cytostatic and/or a cytotoxic agent are usedfor treating a patient with a cancer. A useful class of cytotoxic orcytostatic agents which may be conjugated to an antibody of theinvention, include, but are not limited to, the following non-mutuallyexclusive classes of agents: alkylating agents, anthracyclines,antibiotics, antifolates, antimetabolites, antitubulin agents,auristatins, chemotherapy sensitizers, DNA minor groove binders, DNAreplication inhibitors, duocarmycins, etoposides, fluorinatedpyrimidines, lexitropsins, nitrosoureas, platinols, purineantimetabolites, puromycins, radiation sensitizers, steroids, taxanes,topoisomerase inhibitors, and vinca alkaloids.

Individual cytotoxic or cytostatic agents encompassed by the inventioninclude but are not limited to an androgen, anthramycin (AMC),asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan,buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU),CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide,cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine,dactinomycin (formerly actinomycin), daunorubicin, decarbazine,docetaxel, doxorubicin, estrogen, 5-fluordeoxyuridine, 5-fluorouracil,gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine(CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate,mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel,plicamycin, procarbizine, streptozotocin, tenoposide, 6-thioguanine,thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16 andVM-26.

In a preferred embodiment, a cytotoxic or cytostatic agent is anantimetabolite. The antimetabolite can be a purine antagonist (e.g.,azothioprine or mycophenolate mofetil), a dihydrofolate reductaseinhibitor (e.g., methotrexate), acyclovir, gangcyclovir, zidovudine,vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine,dideoxyuridine, iododeoxyuridine, poscamet, and trifluridine.

Techniques for conjugating such therapeutic moieties to proteins, and inparticular to antibodies, are well known, see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies ForDrug Delivery”, in Controlled Drug Delivery (2nd ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc., 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

5.6.4. Targeted Disruption of SGA-56M and/or SGA-56Mv Expression

As briefly described in Section 5.2.4, supra, endogenous SGA-56M and/orSGA-56Mv gene expression can also be reduced by inactivating or“knocking out” the gene or its promoter using targeted homologousrecombination. (e.g., see Smithies et al., 1985, Nature 317:230; Thomas& Capecchi, 1987, Cell 51:503; Thompson et al., 1989 Cell 5:313). Forexample, a mutant, non-functional SGA-56M and/or SGA-56Mv gene flankedby DNA homologous to the endogenous SGA-56M and/or SGA-56Mv gene (eitherthe coding regions or regulatory regions of the SGA-56M and/or SGA-56Mvgene) can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express SGA-56M and/orSGA-56Mv in vivo. Insertion of the DNA construct, via targetedhomologous recombination, results in inactivation of the SGA-56M and/orSGA-56Mv gene. Such approaches are particularly suited wheremodifications to ES (embryonic stem) cells can be used to generateanimal offspring with an inactive SGA-56Mand/or SGA-56Mv gene homolog(e.g., see Thomas & Capecchi 1987 supra and Thompson 1989, supra). Suchtechniques can also be utilized to generate animal models of breastcancer and other types of cancer. It should be noted that this approachcan be adapted for use in humans provided the recombinant DNA constructsare directly administered or targeted to the required site in vivo usingappropriate vectors, e.g., herpes virus vectors, retrovirus vectors,adenovirus vectors, or adeno associated virus vectors.

Alternatively, endogenous SGA-56M and/or SGA-56Mv gene expression can bereduced by targeting deoxyribonucleotide sequences complementary to theregulatory region of the SGA-56M and/or SGA-56Mv genes (i.e., theSGA-56M and/or SGA-56Mv gene promoter and/or enhancers) to form triplehelical structures that prevent transcription of the SGA-56M and/orSGA-56Mv gene in target cells in the body. (See generally, Helene, 1991,Anticancer Drug Des. 6(6):569; Helene et al., 1992, Ann, N.Y. Acad. Sci.660:27; and Maher, 1992, Bioassays 14(12):807).

5.6.5. Combination Therapies

The administration of an SGA-56M and/or SGA-56Mv antagonist may be usedin conjunction with an anti-cancer agent to potentiate the effect ofeither or both the anti-cancer agent(s) and the antagonist. In apreferred embodiment, the invention further encompasses the use ofcombination therapy to prevent or treat cancer. In one embodiment, anSGA-56M and/or SGA-56Mv antagonist antagonizes (i.e., reduces orinhibits) SGA-56M and/or SGA-56Mv expression or activity. In yet anotherembodiment, the SGA-56M and/or SGA-56Mv antagonist reduces or inhibitseither SGA-56M and/or SGA-56Mv expression or activity.

In one embodiment, breast cancer and other cancers (e.g., ovarian,lymphoid or skin cancer) can be treated with a pharmaceuticalcomposition comprising an SGA-56M and/or SGA-56Mv antagonist incombination with, for example, 5-fluorouracil, cisplatin, docetaxel,doxorubicin, Herceptin®, gemcitabine (Seidman, 2001, Oncology 15:11-14),IL-2, paclitaxel, and/or VP-16 (etoposide).

Such combination therapies may also be used to prevent cancer, preventthe recurrence of cancer, or prevent the spread or metastasis of acancer.

Combination therapy also includes, in addition to administration of anSGA-56M and/or SGA-56Mv antagonist, the use of one or more molecules,compounds or treatments that aid in the prevention or treatment ofcancer (i.e., cancer therapeutics), which molecules, compounds ortreatments includes, but is not limited to, chemoagents,immunotherapeutics, cancer vaccines, anti-angiogenic agents, cytokines,hormone therapies, gene therapies, and radiotherapies.

In one embodiment, one or more chemoagents, in addition to an SGA-56Mand/or SGA-56Mv antagonist, is administered to treat a cancer patient. Achemoagent (or “anti-cancer agent” or “anti-tumor agent” or “cancertherapeutic”) refers to any molecule or compound that assists in thetreatment of a cancer. Examples of chemoagents contemplated by thepresent invention include, but are not limited to, cytosine arabinoside,taxoids (e.g., paclitaxel, docetaxel), anti-tubulin agents (e.g.,paclitaxel, docetaxel, epothilone B, or its analogues), macrolides(e.g., rhizoxin) cisplatin, carboplatin, adriamycin, tenoposide,mitozantron, discodermolide, eleutherobine, 2-chlorodeoxyadenosine,alkylating agents (e.g., cyclophosphamide, mechlorethamine, thioepa,chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, thio-tepa),antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,mithramycin, anthramycin), antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, flavopiridol,5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozolamide),asparaginase, Bacillus Calmette and Guerin, diphtheria toxin,hexamethylmelamine, hydroxyurea, LYSODREN®, nucleoside analogues, plantalkaloids (e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan(CAMPTOSAR, CPT-11), vincristine, vinca alkyloids such as vinblastine),podophyllotoxin (including derivatives such as epipodophyllotoxin, VP-16(etoposide), VM-26 (teniposide)), cytochalasin B, colchine, gramicidinD, ethidium bromide, emetine, mitomycin, procarbazine, mechlorethamine,anthracyclines (e.g., daunorubicin (formerly daunomycin), doxorubicin,doxorubicin liposomal), dihydroxyanthracindione, mitoxantrone,mithramycin, actinomycin D, procaine, tetracaine, lidocaine,propranolol, puromycin, anti-mitotic agents, abrin, ricin A, pseudomonasexotoxin, nerve growth factor, platelet derived growth factor, tissueplasminogen activator, aldesleukin, allutamine, anastrozle,bicalutamide, biaomycin, busulfan, capecitabine, carboplain,chlorabusil, cladribine, cylarabine, daclinomycin, estramusine,floxuridhe, gamcitabine, gosereine, idarubicin, itosfamide, lauprolideacetate, levamisole, lomusline, mechlorethamine, magestrol, acetate,mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin, picamycin,riuxlmab, campath-1, straplozocin, thioguanine, tretinoin, vinorelbine,or any fragments, family members, or derivatives thereof, includingpharmaceutically acceptable salts thereof. Compositions comprising oneor more chemoagents (e.g., FLAG, CHOP) are also contemplated by thepresent invention. FLAG comprises fludarabine, cytosine arabinoside(Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine,doxorubicin, and prednisone.

In one embodiment, said chemoagent is gemcitabine at a dose ranging from100 to 1000 mg/m²/cycle. In one embodiment, said chemoagent isdacarbazine at a dose ranging from 200 to 4000 mg/m² cycle. In apreferred embodiment, said dose ranges from 700 to 1000 mg/m²/cycle. Inanother embodiment, said chemoagent is fludarabine at a dose rangingfrom 25 to 50 mg/m²/cycle. In another embodiment, said chemoagent iscytosine arabinoside (Ara-C) at a dose ranging from 200 to 2000mg/m²/cycle. In another embodiment, said chemoagent is docetaxel at adose ranging from 1.5 to 7.5 mg/kg/cycle. In another embodiment, saidchemoagent is paclitaxel at a dose ranging from 5 to 15 mg/kg/cycle. Inyet another embodiment, said chemoagent is cisplatin at a dose rangingfrom 5 to 20 mg/kg/cycle. In yet another embodiment, said chemoagent is5-fluorouracil at a dose ranging from 5 to 20 mg/kg/cycle. In yetanother embodiment, said chemoagent is doxorubicin at a dose rangingfrom 2 to 8 mg/kg/cycle. In yet another embodiment, said chemoagent isepipodophyllotoxin at a dose ranging from 40 to 160 mg/kg/cycle. In yetanother embodiment, said chemoagent is cyclophosphamide at a doseranging from 50 to 200 mg/kg/cycle. In yet another embodiment, saidchemoagent is irinotecan at a dose ranging from 50 to 150 mg/m²/cycle.In yet another embodiment, said chemoagent is vinblastine at a doseranging from 3.7 to 18.5 mg/m²/cycle. In yet another embodiment, saidchemoagent is vincristine at a dose ranging from 0.7 to 2 mg/m²/cycle.In yet another embodiment, said chemoagent is methotrexate at a doseranging from 3.3 to 1000 mg/m²/cycle.

In a preferred embodiment, the invention further encompasses the use oflow doses of chemoagents when administered in conjunction with anSGA-56M and/or SGA-56Mv antagonist in a combination treatment regimen.For example, initial treatment with an SGA-56M and/or SGA-56Mvantagonist increases the sensitivity of a tumor to subsequent challengewith a dose of chemoagent, which dose is near or below the lower rangeof dosages when the chemoagent is administered in the absence of anSGA-56M and/or SGA-56Mv antagonist. In one embodiment, an SGA-56M and/orSGA-56Mv antagonist and a low dose (e.g., 6 to 60 mg/m²/day or less) ofdocetaxel are administered to a cancer patient. In another embodiment,an SGA-56M and/or SGA-56Mv antagonist and a low dose (e.g., 10 to 135mg/m²/day or less) of paclitaxel are administered to a cancer patient.In yet another embodiment, an SGA-56M and/or SGA-56Mv antagonist and alow dose (e.g., 2.5 to 25 mg/m²/day or less) of fludarabine areadministered to a cancer patient. In yet another embodiment, an SGA-56Mand/or SGA-56Mv antagonist and a low dose (e.g., 0.5 to 1.5 g/m²/day orless) of cytosine arabinoside (Ara-C) are administered to a cancerpatient.

The invention, therefore, contemplates the use of one or more SGA-56Mand/or SGA-56Mv antagonists, which is administered prior to,subsequently, or concurrently with low doses of chemoagents, for theprevention or treatment of cancer.

In one embodiment, said chemoagent is gemcitabine at a dose ranging from10 to 100 mg/m²/cycle.

In one embodiment, said chemoagent is cisplatin, e.g., PLATINOL™ orPLATINOL-AQ™(Bristol Myers), at a dose ranging from 5 to 75 mg/m²/cycle.In another embodiment, a dose of cisplatin ranging from 7.5 to 75mg/m²/cycle is administered to a patient with ovarian cancer or othercancer. In another embodiment, a dose of cisplatin ranging from 5 to 50mg/m²/cycle is administered to a patient with bladder cancer or othercancer.

In another embodiment, said chemoagent is carboplatin, e.g.,PARAPLATIN™(Bristol Myers), at a dose ranging from 2 to 75 mg/m²/cycle.In another embodiment, a dose of carboplatin ranging from 7.5 to 75mg/m²/cycle is administered to a patient with ovarian cancer or othercancer. In another embodiment, a dose of carboplatin ranging from 5 to50 mg/m²/cycle is administered to a patient with bladder cancer or othercancer. In another embodiment, a dose of carboplatin ranging from 2 to20 mg/m²/cycle is administered to a patient with testicular cancer orother cancer.

In another embodiment, said chemoagent is docetaxel, e.g., TAXOTERE™(Rhone Poulenc Rorer) at a dose ranging from 6 to 60 mg/m²/cycle.

In another embodiment, said chemoagent is paclitaxel, e.g., TAXOL™(Bristol Myers Squibb), at a dose ranging from 10 to 135 mg/kg/cycle.

In another embodiment, said chemoagent is 5-fluorouracil at a doseranging from 0.5 to 5 mg/kg/cycle.

In another embodiment, said chemoagent is doxorubicin, e.g., ADRIAMYCIN™(Pharmacia & Upjohn), DOXIL (Alza), RUBEX™ (Bristol Myers Squibb), at adose ranging from 2 to 60 mg/kg/cycle.

In another embodiment, an SGA-56M and/or SGA-56Mv antagonist isadministered in combination with one or more immunotherapeutic agents,such as antibodies or immunomodulators, which include, but are notlimited to, Herceptin®, Retuxan®, OvaRex, Panorex, BEC2, IMC-C225,Vitaxin, Campath I/H, Smart MI95, LymphoCide, Smart I D10, and Oncolym,rituxan, rituximab, gemtuzumab, or trastuzumab.

In another embodiment, an SGA-56M and/or SGA-56Mv antagonist isadministered in combination with one or more anti-angiogenic agents,which includes, but is not limited to, angiostatin, thalidomide, kringle5, endostatin, Serpin (Serine Protease Inhibitor) anti-thrombin, 29 kDaN-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin,16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragmentof platelet factor-4, a β-amino acid peptide corresponding to a fragmentof platelet factor-4 (Maione et al., 1990, Cancer Res. 51:2077), a14-amino acid peptide corresponding to a fragment of collagen I (Tolmaet al., 1993, J. Cell Biol. 122:497), a 19 amino acid peptidecorresponding to a fragment of Thrombospondin I (Tolsma et al., 1993, J.Cell Biol. 122:497), a 20-amino acid peptide corresponding to a fragmentof SPARC (Sage et al., 1995, J. Cell. Biochem. 57:1329-), or anyfragments, family members, or derivatives thereof, includingpharmaceutically acceptable salts thereof.

Other peptides that inhibit angiogenesis and correspond to fragments oflaminin, fibronectin, procollagen, and EGF have also been described (Seethe review by Cao, 1998, Prog. Mol. Subcell. Biol. 20:161). Monoclonalantibodies and cyclic pentapeptides, which block certain integrins thatbind RGD proteins (i.e., possess the peptide motif Arg-Gly-Asp), havebeen demonstrated to have anti-vascularization activities (Brooks etal., 1994, Science 264:569; Hammes et al., 1996, Nature Medicine 2:529).Moreover, inhibition of the urokinase plasminogen activator receptor byantagonists inhibits angiogenesis, tumor growth and metastasis (Min etal., 1996, Cancer Res. 56:2428-33; Crowley et al., 1993, Proc Natl Acad.Sci. USA 90:5021). Use of such anti-angiogenic agents is alsocontemplated by the present invention.

In another embodiment, an SGA-56M and/or SGA-56Mv antagonist isadministered in combination with a regimen of radiation.

In another embodiment, an SGA-56M and/or SGA-56Mv antagonist isadministered in combination with one or more cytokines, which includes,but is not limited to, lymphokines, tumor necrosis factors, tumornecrosis factor-like cytokines, lymphotoxin-α, lymphotoxin-β,interferon-β, interferon-β, macrophage inflammatory proteins,granulocyte monocyte colony stimulating factor, interleukins (including,but not limited to, interleukin-1, interleukin-2, interleukin-6,interleukin-12, interleukin-15, interleukin-18), OX40, CD27, CD30, CD40or CD137 ligands, Fas-Pas ligand, 4-1BBL, endothelial monocyteactivating protein or any fragments, family members, or derivativesthereof, including pharmaceutically acceptable salts thereof.

In yet another embodiment, an SGA-56M and/or SGA-56Mv antagonist isadministered in combination with a cancer vaccine. Examples of cancervaccines include, but are not limited to, autologous cells or tissues,non-autologous cells or tissues, carcinoembryonic antigen,alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine,melanocyte lineage proteins (e.g., gp100, MART-1/MelanA, TRP-1 (gp75),tyrosinase, widely shared tumor-associated, including tumor-specific,antigens (e.g., BAGE, GAGE-1, GAGE-2, MAGE-1, MAGE-3,N-acetylglucosaminyltransferase-V, p15), mutated antigens that aretumor-associated (β-catenin, MUM-1, CDK4), nonmelanoma antigens (e.g.,HER-2/neu (breast and ovarian carcinoma), human papillomavirus-E6, E7(cervical carcinoma), MUC-1 (breast, ovarian and pancreatic carcinoma).For human tumor antigens recognized by T-cells, see generally Robbinsand Kawakami, 1996, Curr. Opin. Immunol. 8:628. Cancer vaccines may ormay not be purified preparations.

In yet another embodiment, an SGA-56M and/or SGA-56Mv antagonist is usedin association with a hormonal treatment. Hormonal therapeutictreatments comprise hormonal agonists, hormonal antagonists (e.g.,flutamide, tamoxifen, leuprolide acetate (LUPRON), LH-RH antagonists),inhibitors of hormone biosynthesis and processing, and steroids (e.g.,dexamethasone, retinoids, betamethasone, cortisol, cortisone,prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids,estrogen, testosterone, progestins), antigestagens (e.g., mifepristone,onapristone), and antiandrogens (e.g., cyproterone acetate).

In yet another embodiment, an SGA-56M and/or SGA-56Mv antagonist is usedin association with a gene therapy program in the treatment of cancer.In one embodiment, gene therapy with recombinant cells secretinginterleukin-2 is administered in combination with an SGA-56M and/orSGA-56Mv antagonist to prevent or treat cancer, particularly breastcancer (See, e.g., Deshmukh et al., 2001, J. Neurosurg. 94:287).

In one embodiment, an SGA-56M and/or SGA-56Mv antagonist isadministered, in combination with at least one cancer therapeutic agent,to a cancer patient for a short treatment cycle to ameliorate thesymptoms of the cancer and potentially eliminate the cancer. Theduration of treatment with the cancer therapeutic agent may varyaccording to the particular cancer therapeutic agent used. The inventionalso contemplates discontinuous administration or daily doses dividedinto several partial administrations. Appropriate treatment time-linesfor cancer therapeutic agents will be appreciated by those skilled inthe art, and the invention contemplates the continued assessment ofoptimal treatment schedules for each cancer therapeutic agent.

The present invention contemplates at least one cycle, preferably morethan one cycle during which a single therapeutic or sequence oftherapeutics is administered. An appropriate period of time for onecycle will be appreciated by the skilled artisan, as will the totalnumber of cycles, and the interval between cycles. The inventioncontemplates the continued assessment of optimal treatment schedules foreach SGA-56M and/or SGA-56Mv antagonist and cancer therapeutic agent.

5.7. Pharmaceutical Preparations and Methods of Administration

The compounds, proteins, peptides, nucleic acid sequences and fragmentsthereof, described herein can be administered to a patient attherapeutically effective doses to treat cancer, e.g., breast cancerwherein the expression level of the SGA-56M and/or SGA-56Mv gene iselevated compared to a non-cancerous sample or a predeterminednon-cancerous standard. A therapeutically effective dose refers to thatamount of a compound sufficient to result in a healthful benefit in thetreated subject.

5.7.1. Effective Dose

Toxicity and therapeutic efficacy of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage tounaffected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured by any technique known in the art, for example, by highperformance liquid chromatography.

5.7.2. Formulations and Use

The invention relates to pharmaceutical compositions, including, but notlimited to pharmaceutical compositions comprising an SGA-56M and/orSGA-56Mv gene product, or antagonists thereof, for the treatment orprevention of cancer.

Pharmaceutical compositions for use in accordance with the presentinvention, e.g., methods to treat or prevent cancer, can be formulatedin a conventional manner using one or more physiologically acceptablecarriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvents can be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g. sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration (i.e.,intravenous or intramuscular) by injection, via, for example, bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

5.8. Vaccine Therapy

Peptides and proteins encoded by the SGA-56M or SGA-56Mv gene andnucleic acids which encode an SGA-56M or SGA-56Mv polypeptide orfragments thereof, can be used as vaccines by administering to anindividual at risk of developing cancer an amount of said protein,peptide, or nucleic acid that effectively stimulates an immune responseagainst an SGA-56M or SGA-56Mv-encoded polypeptide and protects thatindividual from cancer. The invention thus contemplates a method ofvaccinating a subject against cancer wherein said subject at risk fordeveloping a cancer.

Many methods may be used to introduce the vaccine formulations describedabove, these include but are not limited to intranasal, intratracheal,oral, intradermal, intramuscular, intraperitoneal, intravenous, andsubcutaneous routes. Various adjuvants may be used to increase theimmunological response, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art.

The nucleotides of the invention, including variants and derivatives,can be used as vaccines, e.g., by genetic immunization. Geneticimmunization is particularly advantageous as it stimulates a cytotoxicT-cell response but does not utilize live attenuated vaccines, which canrevert to a virulent form and infect the host causing complications frominfection. As used herein, genetic immunization comprises inserting thenucleotides of the invention into a host, and that the nucleotide uptakeby the host cells and the proteins encoded by the nucleotides aretranslated. These translated proteins are then either secreted orprocessed by the host cell for presentation to immune cells and animmune reaction is stimulated. Preferably, the immune reaction is acytotoxic T cell response, however, a humoral response or macrophagestimulation is also useful in preventing initial or additional tumorgrowth and metastasis or spread of a cancer. The skilled artisan willappreciate that there are various methods for introducing foreignnucleotides into a host animal and subsequently into cells for geneticimmunization, for example, by intramuscular injection of about 50 mg ofplasmid DNA encoding the proteins of the invention solubilized in 50 mlof sterile saline solution, with a suitable adjuvant (See, e.g., Weinerand Kennedy, 1999, Scientific American 7:50-57; Lowrie et al., 1999,Nature 400:269-271).

The invention thus provides a vaccine formulation for the preventionand/or treatment of cancer comprising an immunogenic amount of anSGA-56M or SGA-56Mv gene product. The invention further provides for animmunogenic composition comprising a purified SGA-56M or SGA-56Mv geneproduct.

5.9. Kits

The invention includes a kit for assessing the presence of cancer cellsincluding breast cancer cells (e.g., in a sample such as a patientsample). The kit comprises a plurality of reagents, each of which iscapable of binding specifically with a nucleic acid or polypeptidecorresponding to a marker of the invention, e.g., the SGA-56M orSGA-56Mv gene or gene product or fragment thereof. Suitable reagents forbinding with a polypeptide corresponding to a marker of the inventioninclude antibodies, antibody derivatives, labeled antibodies, antibodyfragments, and the like. Suitable reagents for binding with a nucleicacid (e.g., a genomic DNA, an mRNA, a spliced mRNA, a cDNA, or the like)include complementary nucleic acids. For example, the nucleic acidreagents may include oligonucleotides (labeled or non-labeled) fixed toa substrate, labeled oligonucleotides not bound with a substrate, pairsof PCR primers, molecular beacon probes, and the like.

The kit of the invention may optionally comprise additional componentsuseful for performing the methods of the invention. By way of example,the kit may comprise fluids (e.g., SSC buffer) suitable for annealingcomplementary nucleic acids or for binding an antibody with a proteinwith which it specifically binds, one or more sample compartments, aninstructional material which describes performance of a method of theinvention, a sample of normal cells, a sample of cancer cells, and thelike.

6. EXAMPLES

The isolation of novel breast cancer-associated antigens SGA-56M andSGA-56Mv (Seattle Genetics Antigen isolated from MCF-7 cells, andvariants (v) thereof) is described. The nucleic acid sequences ofSGA-56M (SEQ ID NO: 1) and SGA-56Mv (SEQ ID NO: 3) have been depositedwith GenBank. MCF-7 is an estrogen receptor positive (ER+) breastadenocarcinoma cell-line. Suppression Subtractive Hybridization (SSH)and high-throughput cDNA microarrays were combined in analyzing genesover-expressed in breast cancer. The results detail the effectiveness ofcombining SSH and cDNA microarrays in providing breast cancer-specificexpression profiles. Sequence analysis revealed heretoforeuncharacterized molecules SGA-56M and SGA-56Mv, and several previouslyidentified cancer-specific genes. The SGA-56M cDNA (FIG. 1) and SGA-56MvcDNA (FIG. 2) were cloned by PCR and sequence verified by automatedfluorescent sequencing (Applied Biosystems, Foster City, Calif.). Basedon their tumor selectivity (described in Section 6.3), SGA-56M andSGA-56Mv are useful therapeutic targets and/or diagnostic markers in thetreatment of breast cancer, lung cancer, and other SGA-56M or SGA-56Mvpositive cancers.

6.1. Introduction

Breast cancer arises from a malignancy of epithelial cells in thefemale, and occasionally the male, usually of adenocarcinomal origininitiated in the ductal breast epithelium. The majority of breast cancercases are estrogen-dependent adenocarcinomas. The MCF-7 breastcancer-derived tumor cell line is an estrogen-dependent example. BreastCancer is the most common non-dermal malignancy in women and 192,200cases are anticipated in the U.S. for the upcoming year. Despite recentadvances in early diagnosis and treatment, 40,200 U.S. women havesuccumbed to this disease in the year 2000 (Greenlee et al., 2001,Cancer Statistics 51(1): 15). Breast cancer, second only to lung cancerin mortality rates annually, requires continued discovery of additionaluncharacterized antigens and innovative utility of these molecules toimprove overall therapy and intervention.

In total, 10% of all breast cancers are initiated by a genetic mutationsimilar to BRCA-1 and BRCA-2 (Nathanson et al., 2001, Nature Med. 7(5):552). The transformation of normal epithelium and progression tometastatic breast cancer arises from a cascade of genetic alterationsthat translate to global changes in cellular protein composition andexpression. Some of these changes, detected in the form of cell-surfacemarkers, are the focus of present diagnostic and tumor targetingefforts. For example, the HER-2/neu oncogene, which encodes a 185-kDaprotein transmembrane protein, is overexpressed in 10-30% of invasivebreast cancers, 40-60% of intraductal breast carcinomas, as well asother cancer types (Koeppen et al., 2001, Histopathology 38(2): 96).Antibodies to HER2-neu (Herceptin®) have been shown to identify andselectively sensitize antigen positive cells to anti-cancer therapy(Baselga et al., 1998, Cancer Res. 58:2825).

The sex steroid estrogen has been shown to play a major role in tissuedevelopment as well as other physiological processes. In addition, ithas been reported to play a critical role in the progression of bothbreast and gynecological cancers (Pike et al., 1993, Epidemiol. Rev.15:17). MCF-7 is a well-established tumor cell-line that is an ER+adenocarcinoma. Despite its existence in cell-culture for nearly threedecades, it remains likely that many durable alterations in geneexpression patterns still persist since its isolation and initialcharacterization in 1973 (Brooks et al., 1973, J. Biol. Chem. 248(17):6251). Some of the stable genes, and specifically SGA-56M or SGA-56Mv asdescribed herein provide potential targets for diagnostic or therapeuticstrategies for breast cancer.

To evaluate the hypothesis that many of such targets have remainedunrecognized, tumor-enriched SSH libraries were constructed and arrayedto selectively screen for tumor-specific genes. SSH is a technique wellknown in the art for its effectiveness in characterizing andprioritizing differentially expressed genes: (Chu et al., 1997, Proc.Natl. Acad. Sci 94(19): 10057; Gurskaya et al., 1996, Anal. Biochem.240: 90; Kuang et al., 1998, Nuc. Acid Res. 26: 1116; von Stein et al.,1997, Nuc. Acid Res. 25: 2598; Wong et al., 1997, J. Biol. Chem.272(40): 25190; and Yokomizo et al., 1997, Nature 387: 620). SGA-56M orSGA-56Mv, novel breast cancer-associated proteins, were discoveredutilizing these techniques as described herein. The initialtumor-enriched MCF-7-specific SSH libraries were evaluated in a higherdensity format with minimal redundancy, demonstrating that the overallcomplexity of the libraries had not been compromised.

Intensive and systematic evaluation of gene expression patterns iscrucial in understanding the physiological mechanisms associated withcellular transformation and metastasis. Currently, several technicalplatforms are being used to accomplish this goal. They include: SerialAnalysis of Gene Expression (SAGE) (Velculescu et al., 1995, Science270: 484), Restriction Enzyme Analysis of Differentially ExpressedSequences (READS) (Prasher et al., 1999, Methods Enzymol. 303: 258),Amplified Fragment Length Polymorphism (AFLP) (Bachem et al., 1996,Plant J. 2: 745), Representational Difference Analysis (RDA) (Hubank etal., 1994, Nucleic Acid Res. 22(25): 5640), Differential Display (Lianget al, 1992, Cancer Res. 52(24): 6966) and SSH (Diatchenko et al., 1996,Proc. Natl. Acad. Sci. 93: 6025) as detailed in this text. SSH is verysimilar to RDA with the exception of an additional normalization stepthat is included to increase the relative abundance of rare transcripts.The combination of SSH and cDNA microarrays offers several advantagesover the aforementioned technologies in the discovery of noveltumor-associated proteins and antigens (TAA's). The use of SSH is anattractive approach to identifying novel cancer targets because it doesnot rely on previously characterized cDNA sets. SSH efficientlynormalizes both frequent and rare transcripts at equivalent levels andpreferentially amplifies only those which are differentially expressed.The use of expression arrays further increases the chances ofidentifying lead targets by examining thousands of genes in a singleexperiment.

6.2. Materials and Methods

6.2.1. Cell Culture

Breast tumor cell-lines MCF-7, T47-D, SKBR-3, MDA-MB-231, MDA-MB-435s,MDA-MB-453, H3396, Hs578T and BT-549 were grown in RPM 1640 medium®supplemented with 10% fetal bovine serum plus 100 U/mL penicillin G and100 μg/mL streptomycin sulfate. All tumor cell-lines were passaged onceper week by trypsinization and replated at 2500-3000 cells/cm². Normalhuman mammary epithelial cells (HMEC) were maintained in MEGM®(Clonetics, San Diego, Calif.). HMEC's were passaged once per week bytrypsinization and replating at 2500-3000 cells/cm².

6.2.2. RNA Isolation

Total RNA was isolated from cultured cells using RNA-Bee™ (Tel-Test,Inc., Friendswood, Tex.). Poly A+ RNA was extracted using the OligotexmRNA Midi kit® (Qiagen, Inc., Valencia, Calif.).

6.23. Generation of SSH cDNA Libraries

MCF-7 breast cancer-specific SSH cDNA libraries were constructedessentially as described by Diatchenko et al., 1996, Proc. Natl. Acad.Sci. 93:6025. Library one was constructed using the breast tumor ER+cell-line MCF-7 (tester) vs. HMEC (driver). Library two was constructedusing the breast tumor ER+ cell-line MCF-7 (tester) vs. a pool of 5 ER−cell lines (SKBR-3, MDA-MB-231, MDA-MB435s, Hs578T, and BT-549)(driver).

Driver cDNA was synthesized from 2 ug of poly A+ RNA using 1 ul of 10 uMcDNA synthesis primer 5′-TTTTGTACAAGCTT₃₀N₁N-3′ (SEQ ID NO: 7) and 1 ulof 200 u/ul Superscript II Reverse Transcriptase® (Invitrogen, Carlsbad,Calif.). The resulting cDNA pellet was digested with 1.5 ul of 10u/ul ofRsa I restriction enzyme. Driver cDNA's were then precipitated with 100ul of 10M Ammonium Acetate (Sigma, St. Louis, Mo.), 3 ul of 20 mg/mlglycogen (Roche Molecular Biochemicals, Indianapolis, Ind.) and 1 ml ofethanol (Sigma, St. Louis, Mo.). The cDNA preparations were thenresuspended in 5 ul of diethyl pyrocarbonate (DEPC) treated water.

Tester cDNA was synthesized from 2 ug of poly A+ RNA as described abovefor the driver. Rsa I digested tester cDNA was diluted in 5 ul of DEPCtreated water prior to adaptor ligation. Diluted tester cDNA (2 ul) wasligated to 2 ul of 10 uM adaptor 1(5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′) (SEQ ID NO: 8) and2 ul of 10 uM adaptor2R(S-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3′) (SEQ ID NO: 9) inseparate reactions using 0.5 units of T4 DNA ligase (Invitrogen,Carlsbad, Calif.).

Driver cDNA (600 ng) was added separately to each of the two tubescontaining adaptor-1 ligated tester (20 ng) and adaptor 2R ligatedtester (20 ng). The samples were mixed, ethanol precipitated asdescribed above, and resuspended in 1.5 ul of hybridization buffer (50mM Hepes pH 8.3, 0.5 M NaCl/0.0.2 mM EDTA pH 8.0). The reaction mixturewas placed in hot start PCR tubes, (Molecular BioProducts, San Diego,Calif.), denatured at 95° C. for 1.5 min. and then incubated at 68° C.for 8 hrs. After this initial hybridization, the samples were combinedand excess heat denatured driver cDNA (150 ng) was added. This secondaryreaction mixture was incubated overnight at 68° C. The finalhybridization mixture was diluted in 200 ul of dilution buffer (20 mMHepes pH 8.3, 50 mM NaCl, 0.2 mM EDTA) and stored at −20° C.

Two rounds of PCR amplification were performed for each SSH library. Theprimary PCR was performed in 25 ul. The reaction mixture contained 1 ulof diluted subtracted cDNA, 1 ul of 10 uM PCR primer 1(5′-CTAATACGACTCACTATAGGGC-3′) (SEQ ID NO: 10), 10× PCR bufferconsisting of (166 mM NH₄C₂H₃O₂, 670 mM Tris pH 8.8, 67 mM MgCl₂, and100 mM 2-Mercaptoethanol), 1.5 ul of 10 mM dNTP's, 1.5 ul DimethylSulfoxide (DMSO) (Sigma, St Louis, Mo.), and 0.25 ul of 5 u/ul of Taqpolymerase (Brinkmann, Westbury, N.Y.). PCR was performed with thefollowing cycling conditions: 75° C. for 7 min.; 94° C. for 2 min.; 27cycles at 94° C. for 30 sec., 66° C. for 30 sec., and 72° C. for 1.5min.; and a final extension at 72° C. for 5 min. A secondary PCR wasperformed using 1 ul of the primary PCR as template with the samereaction components as above. Nested PCR primers NP1(5′-TCGAGCGGCCGCCCGGGCAGGT-3′) (SEQ ID NO: 11) and NP2R(5′-AGCGTGGTCGCGGCCGAGGT-3′) (SEQ ID NO: 12) were used in place of PCRprimer 1. The secondary PCR was performed with the following cyclingconditions: 94° C. for 2 min.; 15 cycles at 94° C. for 30 sec., 68° C.for 30 sec., and 72° C. for 1.5 min.; and a final extension at 72° C.for 5 min. The PCR products were analyzed on 1.5% ultrapure agarose gels(Invitrogen, Carlsbad, Calif.) and visualized by ethidium bromide(Fisher Chemical, Fair Lawn, N.J.).

Subtraction efficiency was confirmed by PCR depletion of EF-1 andTubulin. EF-1 primers were EF-1 (5′-CTGTTCCTGTTGGCCGAGTC-3′) (SEQ ID NO:13) and EF-2 (5′-CGATGCATTGTTATCATTAAC-3′) (SEQ ID NO: 14). Tubulinprimers were Tu-1 (5′-CACCCTGAGCAGCTCATCAC-3′) (SEQ ID NO: 15) and Tu2(5′-GGCCAGGGTCACATTTCACC-3′) (SEQ ID NO: 16).

6.2.4. Cloning of SSH Pools into pCR4-TOPO

The SSH-cDNA pools were cloned into the pCR4-TOPO® vector (Invitrogen,Carlsbad, Calif.) and transformed into chemically competent TOP 10cells® (Invitrogen, Carlsbad, Calif.). The library was plated on LB agarplates (Becton Dickinson, Sparks, Md.) containing 50 μg/ul kanamycin(Sigma, St. Louis, Mo.). Cloning efficiency and size distribution foreach library was determined by amplification using M13 (−20)(5′-GTAAAACGACGGCCAGT-3′) (SEQ ID NO: 17) and M1 3R(5′-CAGGAAACAGCTATGACC-3′) (SEQ ID NO: 18) universal primers.

6.2.5. Custom Array Generation

SSH clones containing cDNA sequences of interest were amplified usingM13 (−20) and M13R universal primers. PCR products were purified using96-well MultiScreen PCR Purification Plates (Millipore, Bedford, Mass.).Microarrays were prepared by spotting targets in duplicate on positivelycharged nylon membranes (Hybond-XL®, Amersham Pharmacia Biotech,Piscataway, N.J.) at concentrations of 2 ng DNA/spot using a Biomek 2000Robot® (Beckman Coulter Inc., Fullerton, Calif.). For probeconstruction, mRNA was isolated from cell lines as described above. PolyA+ RNA (1 ug) was converted to cDNA and labeled with (^(α)-P32) dCTP(Amersham Pharmacia Biotech, Piscataway, N.J.) by reverse transcriptionusing Superscript II RT® (Invitrogen, Carlsbad, Calif.). Hybridizationswere performed overnight at 42° C. in 6× Saline Sodium Citrate (SSC),0.1% Sodium Dodecyl Sulfate (SDS), 50% Deionized Formamide, and 5×Denhardt's solution (1% Ficoll Type 400, 1% polyvinylpyrrolidone, and 1%bovine serum albumin) (Research Genetics, Huntsville, Ala.). Washconditions were 4 times in 2×SSC/0.1% SDS for 10 min. each at roomtemperature, followed by 4 high stringency washes in 0.1×SSC/0.1% SDS at65° C. for 30 min. each.

6.2.6. Array Data Analysis

Hybridization Intensities were quantitated on the PhosphorImager SI®(Molecular Dynamics, Sunnyvale, Calif.) using ArrayVision 5.1 Software®(Imaging Research, St. Catharines, ON, CA). Average signal intensitieswere determined for each set of duplicate spots. For each membraneanalyzed, relative quantitative values were determined based onnormalization to multiple housekeeping genes spotted at variouslocations on each membrane. This allowed for blot-to-blot comparisons indetermining differential expression. Two independent microarrayexperiments were performed for each comparison to ensure overallvalidity and reproducibility of the results. Targets greater than 2-foldover-expressed in a tumor vs. normal comparison were considered forfurther evaluation.

6.2.7. Semi-Quantitative RT-PCR

DNA was synthesized from 5 ug total RNA using the SuperscriptFirst-Strand cDNA Synthesis System for RT-PCR® (Invitrogen, Carlsbad,Calif.). Gene specific primers were selected for SGA-56M or SGA-56Mv andEF-1 to obtain semi-quantitative mRNA levels. Primers used were commonfor SGA-56M and SGA-56Mv. They were as follows: RT1(5′-GCTTGGAAAAGTTGAGCC-3′) (SEQ ID NO: 19), and RT2(5′-CTGGGTCTGAGTCTTAGC-3′) (SEQ ID NO: 20). Primers for EF-1 were asfollows: EF-1 (5′-CTGTTCCTGTTGGCCGAGTC-3′) (SEQ ID NO: 13) and EF-2 (5′CGATGCATTGTTATCATTAAC-3′) (SEQ ID NO: 14).

6.2.8. Multiple Tissue Expression Array (MTE™)

The MTE™ (Clontech, Palo Alto, Calif.) array was used to determinerelative expression of SGA-56M/SGA-56Mv in various normal populations.Primers used in amplifying a probe were common for SGA-56M and SGA-56Mv.Primers were as follows: RT1 (5′-GCTTGGAAAAGTTGAGCC-3′) (SEQ ID NO: 19),and RT2 (5′-CTGGGTCTGAGTCTTAGC-3′) (SEQ ID NO: 20). Fifty ng of PCRproduct were labeled using Ready-to-go Beads® (Amersham BiosciencesCorporation, Piscataway, N.J.) and ^(α)-P32 dCTP at 3000 Ci/mmol(Amersham Biosciences Corporation, Piscataway, N.J.). The housekeepingcontrol, EF-1, was used to evaluate the spot-to-spot variability withinthe experiment. See Farkas et al., 2003, J. Biol. Chem. 384: 945 forgrid wherein the positional coordinates of the array are defined.

6.2.9. Cancer Profiling Array (CPA™)

The CPA™ (Clontech, Palo Alto, Calif.) was used to determine theexpression of SGA-56M and SGA-56Mv in numerous tumor/normal pairedpatient samples. The CPA™ contains 241 tumor and adjacent normal pairedpatient isolates. Primers used in amplifying a probe were common forSGA-56M and SGA-56Mv. Primers were as follows: RT1(5′-GCTTGGAAAAGTTGAGCC-3′) (SEQ ID NO: 19), and RT2(5′CTGGGTCTGAGTCTTAGC-3′) (SEQ ID NO; 20). Fifty ng of PCR product waslabeled using Ready-to-go Beads and α-P32 dCTP at 3000 Ci/mmol. A totalof 241 paired cDNA samples were synthesized and spotted onto nylonmembranes for 13 different tumor types. The tumor types included:Breast, Cervix, Colon, Kidney, Lung, Ovarian, Pancreas, Prostate,Rectum, Thyroid Gland, Small Intestine, Stomach, and Uterus. SeeZhumabayeva et al., 2000, BioTechniques. 3: 22 for grid wherein thepositional coordinates of the array are defined.

6.2.10 ABI PRISM®7000 Sequence Detection System

The ABI PRISM® 7000 Real-Time PCR Sequence Detection System (AppliedBiosystems, Foster City, Calif.) was used to determine the breastcancer-selectivity for SGA-56M/SGA-56Mv. The Breast Cancer Rapid-Scan™gene expression RNA panel (OriGene Technologies, Inc., Rockville, Md.)and Lung Cancer patient tumor and adjacent normal tissue RNA (BiochainInstitute, Inc., Hayward, Calif. and Ambion, Inc., Austin, Tex.) wereused in this experiment. The Rapid-Scan™ Panel contains first-strandcDNA derived from 12 tumor/normal breast patient sample pairs. LungCancer cDNA was synthesized from using the Superscript First-Strand cDNASynthesis System for RT-PCR® (Invitrogen, Carlsbad, Calif.). Primers andprobes for SGA-56M and SGA-56Mv were as follows: EXP2-FP(5-TGTCCCAGGAACCTTTCTTCA-3′) (SEQ ID NO: 21), EXP2—RP(5′-CCCAGCTTGCACCTGGTTT-3′) (SEQ ID NO: 22), and EXP2-TaqMan MGB Probe(5′-FAM-CTACAGCTCACTCTCCAG-NFQMGB-3′) (SEQ ID NO: 23). Primers andprobes for EF1 were as follows: EF1-FP (5′-ATGACCCACCAATGGAAGCA-3′) (SEQID NO: 24), EF1-RP (5′-GCCTGGATGGTTCAGGATAATC-3′) (SEQ ID NO: 25), andEF1-TaqMan MGB Probe (5′-VIC-CTGGCTTCACTGCTC-NFQMGB-3′) (SEQ ID NO: 26).EF-1 was used as the normalization gene for all ABI PRISM® 7000experiments.

The Comparative Ct Method (Applied Biosystems, Foster City, Calif.) wasused in calculating tumor vs. normal ratios for SGA-56M/SGA-56Mv. Theamount of target (SGA-56M/SGA-56Mv), normalized to an endogenousreference (EF-1) and relative to a calibrator, is given by thearithmetic formula: 2^(−ΔΔCt) where ΔΔCt is the change in thresholdcycle between target and reference.

6.2.11. Bioinformatics Analysis

After completion of the array data analysis sorting process, interestingnovel targets were retained and analyzed using several additionalcomputational programs. The derived SGA-56M and SGA-56Mv cDNA wasanalyzed using Vector NTI Suite 6.0® (InforMax, Inc., Bethesda, Md.).Transmembrane domain and protein localization analysis were performedusing the ExPASy Proteomics Tools Programs® (Swiss Institute ofBioinformatics, Geneve, Switzerland). Amino acid sequence predictionprograms used included: HMMTOP (Tusnady et al., 1998, J. Mol. Bio.283:489), TM pred (Hofinann et al., 1993, J. Biol. Chem. 347:166), TMHMMv1.0 (Sonnhammer et al., 1998, Proc. of Sixth Int. Conf on IntelligentSystems for Mol. Bio., AAAI Press, pp. 175-182), TMAP, and PSORT (Nakaiet al., 1999, Trends Biochem Sci. 24(1):34).

6.2.12. Subcellular Localization of SGA-56M and SGA-56Mv

The subcellular localization patterns for SGA-56M and SGA-56Mv weredetermined using green fluorescent protein (GFP) reporter constructs.SGA-56M and SGA-56Mv cDNA clones were amplified by PCR usinggene-specific primers: SGA-56M (GFP1)5′-AGCTCTCTCGAGATGTCTTTTCTTGGCATCCTGTGCAAGTGT-3′ (SEQ ID NO: 27) andSGA-56M (GFP2) 5′-AGCTCTAAGCTTTCAGTGTGGAGGGTTCATGGTGCCTTG-3′ (SEQ ID NO:28). Xho I (SGA-56M-GFP1) and Hind III (SGA-56M-GFP2) restriction siteswere included for in-frame cloning (as underlined). The resultingSGA-56M and SGA-56Mv PCR products were restriction digested and clonedinto the Xho I/Hind III-cut pGFP² vector (BioSignal Packard, Montreal,Canada). Expression of this plasmid in eukaryotic cells resulted in thesynthesis of both SGA-56M/GFP and SGA-56Mv/GFP fusion polypeptides.These constructs were transiently transfected into human kidney 293cells, and SKBR-3 breast carcinoma cells by electroporation. Thesubcellular localization patterns for SGA-56M and SGA-56Mv greenfluorescence signals were monitored by fluorescence microscopy.

63 Results

6.3.1. Isolation of the SGA-56M cDNA

The SGA-56M cDNA (FIG. 1) was amplified using gene-specific primers andcloned into the pCR 4.0® TOPO TA vector (Invitrogen, Carlsbad, Calif.).The SGA-56M sequence (FIG. 1) (SEQ ID NO: 1) was sequence verified usingcustom primers (Sigma-Genosys, Woodlands, Tex.) and automatedfluorescent sequencing (PE Applied Biosystems, Foster City, Calif.).

6.3.2. Isolation of the SGA-56Mv cDNA

The SGA-56Mv cDNA (FIG. 2) was amplified using gene-specific primers andcloned into the pCR 4.0® TOPO TA vector (Invitrogen, Carlsbad, Calif.).SGA-56Mv (SEQ ID NO: 3) was identified while screening clones for theisolation of SGA-56M (SEQ ID NO: 1). SGA-56Mv (FIG. 2) was sequenceverified using custom primers (Sigma-Genosys, Woodlands, Tex.) andautomated fluorescent sequencing (PE Applied Biosystems, Foster City,Calif.).

6.3.3. Cancer-Selectivity by Semi-Quantitative PCR

SGA-56M and SGA-56Mv displayed cancer-selectivity on various breastcarcinoma cell-lines (FIG. 3). A cDNA region common to both SGA-56M andSGA-56Mv was amplified in this experiment. All breast cancer cell-linesevaluated were positive for SGA-56M and SGA-56Mv mRNA (FIG. 3). Normalhuman mammary epithelial cells (HMECs) were negative for SGA-56M andSGA-56Mv mRNA expression even after 35 PCR cycles.

SGA-56M and SGA-56Mv displayed positive mRNA expression in other tumorcell-lines (FIG. 4). Positive tumor cell-lines for SGA-56M and SGA-56MvmRNA include: Ramos (Burkitt's lymphoma), NCI-H460 (Non-Small Cell LungCancer, NSCLC), MiaPaCa-2 (Pancreatic Cancer), and WM-115 (Melanoma)(FIG. 4).

6.3.4. Evaluation of Normal Expression by MTE™

SGA-56M and SGA-56Mv mRNA expression levels in normal tissues wereevaluated using the Multiple Tissue Expression (MTE™) Array. A cDNAregion common to both SGA-56M and SGA-56Mv was amplified and used as aprobe for this experiment. The MTE™ Array contains 76 tissue-specificpolyA+ RNA isolates. See Farkas et al., 2003, J. Biol. Chem. 384: 945for a grid of the tissue represented on the array. SGA-56M and SGA-56Mvdisplayed minimal expression on normal tissue. The only significantlevel of normal tissue expression was observed in the testis, atposition 8F (FIG. 5B).

6.3.5. Cancer-Selectivity by CPA™

SGA-56M and SGA-56Mv mRNA cancer-selectivity was evaluated using theCancer Profiling Array (CPA™). A cDNA region common to both SGA-56M andSGA-56Mv was amplified and used as a probe for this experiment. SGA-56Mand SGA-56Mv displayed cancer-selective expression greater than 2-foldin several tumor types (Table 3). Breast tumor and corresponding normaltissue pairs displaying the highest T: N ratios for SGA-56M areillustrated (Table 4). SGA-56M and SGA-56Mv probes were alsocancer-selective in eight patient isolates known to have associatedmetastases. TABLE 3 SGA-56M and SGA-56Mv cancer-selectivity inindividual tumor and corresponding normal tissues >2-fold T:N A. TumorTissues Breast (n = 50) 30% Uterus (n = 42) 19% Colon (n = 35) 14%Stomach (n = 27) 7% Ovary (n = 14) 29% Lung (n = 21) 14% Kidney (n = 20)5% Rectum (n = 18) 33% B. Normal Tissues Breast (n = 50) 0% Uterus (n =42) 2% Colon (n = 35) 0% Stomach (n = 27) 0% Ovary (n = 14) 7% Lung (n =21) 0% Kidney (n = 20) 0% Rectum (n = 18) 0%

TABLE 4 Elevated breast cancer-selectivity of SGA-56M and SGA-56Mv in asubset of patients Tumor type Age T:N Infiltrating ductal carcinoma 52 9Infiltrating ductal carcinoma 45 5 Infiltrating ductal carcinoma 44 5Infiltrating ductal carcinoma 60 4 Infiltrating lobular carcinoma 49 4Infiltrating lobular carcinoma 66 4 Medullary carcinoma 47 3Adenocarcinoma 53 3 Tubular carcinoma 63 3 Fibrosarcoma 44 36.3.6. Cancer-Selectivity by ABI PRISM® 7000 Real-Time PCR

SGA-56M and SGA-56Mv displayed breast cancer-selectivity using thebreast cancer Rapid Scan™ cDNA panel (Table 6). Real-time PCR was usedto further quantify the extent of over-expression of SGA- and 56M andSGA-56Mv in breast and lung patient tumor isolates. Twelve breast tumorand corresponding normal tissues were analyzed for quantitative SGA-56Mand SGA-56Mv expression levels (Table 5). The comparative C_(T) methodwas used in calculating relative quantitative T: N ratios while usingthe control gene EF-1 as a reference. In total, 5 of 12 breast cancerpatient pairs (42%) displayed T: N levels>3-fold (Table 6). A singlecDNA pair (sample #10) displayed a T:N ratio of 14.1 (Table 6).

SGA-56M and SGA-56Mv lung cancer-selectivity was examined using 10 nonsmall-cell lung cancer patient pairs, 5 adenocarcinomas and 5 squamouscell carcinomas with corresponding normal tissues (Table 7).Quantitative T:N ratios were calculated using methods as describedabove. In total, 7 of 10 non small-cell lung cancer patients (70%)displayed T:N levels>3-fold (Table 8). In particular, all of thesquamous cell carcinoma patients appeared to exhibit elevated expressionlevels>4-fold T:N (Table 8). Two cDNA pairs (SQ1 and SQ4) displayed T:Nlevels>10-fold (Table 8). TABLE 5 Histopathology data for breast cancerQPCR tissue panel Sam- ER PR ple Tumor Type ER/PR (fmol/mg) (fmol/mg) 1Invasive mixed tubular ER+/PR+++ 7 233 carcinoma 2 Invasive ductalcarcinoma ER+/PR+++ 14 99 3 Invasive lobular carcinoma ER+++++/ 142 528PR+++++ 4 Invasive ductal carcinoma ER++/PR− 20 9 5 Invasive ductalcarcinoma ER++/PR− 18 7 6 Invasive ductal carcinoma ER+++/PR+ 65 30 7Invasive ductal carcinoma ER++/PR+ 30 32 8 Invasive ductal carcinomaER+/PR− 9 0.5 9 Adenoid cystic carcinoma ER++/PR+ 22 14 10 Invasiveductal carcinoma ER−/PR− 3 0 11 Ductal carcinoma in-situ ER+/PR+ 19 1312 Invasive ductal carcinoma ER+/PR+ 6 26

TABLE 6 SGA-56M and SGA-56Mv breast cancer-selectivity in patient tumorsby QPCR Sample SGA-56M Ct EF-1 Ct ΔCt ΔΔCt T:N Breast Tumor 1 (ER+)31.13 30.35 0.78 −0.97 1.96 Breast Normal 1 30.42 28.67 1.75 BreastTumor 2 (ER+) 31.05 29.61 1.44 −0.58 1.49 Breast Normal 2 30.83 28.812.02 Breast Tumor 3 (ER+) 30.40 29.51 0.89 −2.23 4.70 Breast Normal 331.69 28.57 3.12 Breast Tumor 4 (ER+) 31.18 29.40 1.78 −0.57 1.48 BreastNormal 4 31.82 29.47 2.35 Breast Tumor 5 (ER+) 30.55 27.61 2.94 1.460.36 Breast Normal 5 30.79 29.31 1.48 Breast Tumor 6 (ER+) 31.42 30.820.60 −2.21 4.63 Breast Normal 6 31.08 28.27 2.81 Breast Tumor 7 (ER+)31.57 30.86 0.71 −1.46 2.75 Breast Normal 7 31.78 29.61 2.17 BreastTumor 8 (ER+) 30.80 30.94 −0.14 −1.75 3.36 Breast Normal 8 30.41 28.801.61 Breast Tumor 9 (ER+) 31.26 29.47 1.79 −0.14 1.10 Breast Normal 929.54 27.61 1.93 Breast Tumor 10 (ER−) 30.94 32.33 −1.39 −3.82 14.12Breast Normal 10 30.82 28.39 2.43 Breast Tumor 11 (ER+) 30.92 28.71 2.211.09 0.47 Breast Normal 11 30.07 28.95 1.12 Breast Tumor 12 (ER+) 30.3027.90 2.40 0.81 0.57 Breast Normal 12 30.61 29.02 1.59

TABLE 7 Background information for lung cancer QPCR tissue panel SampleTumor Type Differentiation Age Sex Lung Tumor AD01 AdenocarcinomaModerately Differentiated 44 M Lung Tumor AD02 Adenocarcinoma PoorlyDifferentiated 62 M Lung Tumor AD03 Adenocarcinoma Poorly Differentiated58 F Lung Tumor AD04 Adenocarcinoma Moderately Differentiated 60 M LungTumor AD05 Adenocarcinoma Moderately Differentiated 73 F Lung Tumor SQ01Squamous Cell Carcinoma Well Differentiated 78 M Lung Tumor SQ02Squamous Cell Carcinoma Well Differentiated 62 M Lung Tumor SQ03Squamous Cell Carcinoma Moderately Differentiated 63 F Lung Tumor SQ04Squamous Cell Carcinoma Poorly Differentiated 64 M Lung Tumor SQ05Squamous Cell Carcinoma Moderately Differentiated 66 M

TABLE 8 SGA-56M and SGA-56Mv lung cancer-selectivity in patient tumorsby QPCR Sample SGA-56M Ct EF-1 Ct ΔCt ΔΔCt T:N Lung Tumor AD01 33.4325.91 7.52 −0.45 1.36 Lung Normal 34.08 26.11 7.97 Lung Tumor AD02 33.7826.81 6.97 −2.70 6.50 Lung Normal 35.72 26.05 9.67 Lung Tumor AD03 36.4324.94 11.49 2.00 0.25 Lung Normal 34.62 25.13 9.50 Lung Tumor AD04 36.0525.41 10.64 1.30 0.41 Lung Normal 35.48 26.14 9.34 Lung Tumor AD05 35.2930.24 5.05 −1.74 3.34 Lung Normal 36.48 29.69 6.79 Lung Tumor SQ01 33.7027.60 6.10 −3.48 11.16 Lung Normal 36.10 26.52 9.58 Lung Tumor SQ0235.24 30.12 5.12 −2.72 6.57 Lung Normal 37.04 29.21 7.83 Lung Tumor SQ0332.96 28.35 4.61 −2.84 7.16 Lung Normal 34.84 27.39 7.45 Lung Tumor SQ0434.43 27.41 7.02 −3.73 13.22 Lung Normal 36.92 26.18 10.74 Lung TumorSQ05 37.97 32.07 5.90 −2.03 4.07 Lung Normal 39.25 31.33 7.936.3.7. Sequence Comparison for SGA-56M and SGA-56Mv

Nucleic acid entries sharing homology with SGA-56M include: GenBankAccession No. D87437, GenBank Accession No. NM_(—)014837, GenBankAccession No. AB085674, GenBank Accession No. AX714019, and GenBankAccession No. AX747010.

GenBank Accession No. D87437, and GenBank Accession No. NM_(—)014837 aretermed KIAA0250 in the scientific literature (Nagase et al., 1996, DNAResearch. 3(5): 321-329, 341-354. and Sood et al., 2001, Genomics,73:211-222). KIAA0250 does not share any significant sequence homologyto known motifs that would suggest a potential functional role (Sood etal., 2001, Genomics, 73:211-222). KIAA0250 was identified as 1 of 13novel transcripts that mapped to the hereditary prostate locus (HPC1)(Sood et al., 2001, Genomics, 73:211-222).

GenBank Accession No. AB085674, termed Homo sapiens mRNA SMG-7, alsoshares homology with SGA-56M and SGA-56Mv. To date, no putativebiological role for SMG-7 has been described in the scientificliterature. SMG-1, a novel member of the phosphatidylinositol 3-kinasefamily of proteins, has been reported to be associated withnonsense-mediated mRNA decay (NMD), due presumably to its ability tophosphorylate hUpf1 (Denning et al., 2001, J. Biol. Chem. 276(25):22709-22714). Neither SMG-1 nor SMG-7 has been described as associatedwith cancer.

GenBank Accession No. AX714019 and GenBank Accession No. AX747010correspond to SEQ ID 703 (European Patent Application EP1293569) and SEQID 535 (European Patent Application EP1308459), respectively. Theseapplications disclose molecules (AX714019 and AX747010) that appear tocorrespond to partial cDNA sequences having limited homology to fulllength SGA-56M (SEQ ID NO: 1) and SGA-56Mv (SEQ ID NO: 3). See Table 9.As indicated herein above, SEQ ID NO: 1 and SEQ ID NO: 3 have beendeposited in GenBank. TABLE 9 Alignment Gene Nucleic Acids AX714019 (%)AX747010 (%) SGA-56M 2917 1755/2917 (60%) 1753/2917 (60%) SGA-56Mv 27791617/2779 (58%) 1615/2779 (58%)6.3.8 Subcellular Localization of SGA-56M and SGA-56Mv

Subcellular localization patterns for SGA-56M and SGA-56Mv weredetermined using fluorescence microscopy. Transient expression andsubcellular localization pattern recognition of SGA-56M/GFP andSGA-56Mv/GFP constructs were analyzed using 293 human kidney cells, andSKBR-3 breast carcinoma cells. Expression of GFP alone resulted indiffuse green fluorescence signals throughout the cells. Subcellularlocalization patterns for SGA-56M/GFP and SGA-56Mv/GFP were consistentwith those previously reported for other proteins of cytoplasmic and/orperoxisomal compartments (Simpson et al., 2000, EMBO reports, 3:287-292).

6.4. Discussion

Gene expression profiling provides a systematic approach to studying themechanisms associated with progression from normal to metastaticdisease. In this application, the present inventors have combined SSHand cDNA microarrays to identify the uncharacterized breastcancer-associated antigen, SGA-56M and variants thereof, includingSGA-56Mv. Combining SSH and cDNA microarrays provides a rapid andeffective approach to high-throughput screening for novel tumorassociated antigens (TAAs). The principle of SSH allows for thepreferential amplification of differentially expressed sequences whilesuppressing those present at equal abundance within the initial mRNA(Diatchenko et al., supra). The high level of enrichment, low level ofbackground, and efficient normalization of sequences makes this anattractive approach for the rapid identification of novel targets.SGA-56M cDNA, identified by this method, and variants thereof, includingSGA-56Mv comprise new diagnostic, prognostic, and/or therapeutic targetsfor breast and lung cancer treatment. SGA-56M and SGA-56Mv displaytumor-selective expression in breast and lung cancer, and other cancers,while displaying minimal expression in normal tissues. SGA-56M andSGA-56Mv, based on their elevated level of tumor-selective expression,and association with metastases are strong candidates for considerationand evaluation as a potential mode of intervention for breast cancer andother cancers.

Overall, SGA-56M and SGA-56Mv, can be helpful in providing valuableinsight into the potential mechanisms involved in breast cancerdevelopment and progression. The present inventors have demonstratedthat gene expression profiling studies using SSH and arrays can assistin identifying interesting cancer-selective genes, like SGA-56M andSGA-56Mv, that have not been previously implicated in breast cancer.Studies to investigate further the potential functional role of tumorassociated antigens are extremely helpful in designing experiments tocritically evaluate the pathways and mechanisms necessary for effectivetherapeutic intervention.

7. REFERENCES CITED

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A method for diagnosing cancer in a subject comprising detecting ormeasuring an SGA-56M gene product in a sample derived from said subject,wherein the SGA-56M gene product is: (a) an RNA corresponding to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; (b) a protein comprisingSEQ ID NO:5; (c) a protein comprising SEQ ID NO:6; (d) a nucleic acidcomprising a sequence hybridizable to SEQ ID NO: 1, SEQ ID NO:2, SEQ IDNO:3, or SEQ ID NO:4, or a complement thereof, or a protein comprising asequence encoded by said hybridizable sequence; (e) a nucleic acid atleast 90% homologous to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQID NO:4 or a complement thereof, or a protein encoded thereby; whereindetecting or measuring elevated levels of the SGA-56M gene productrelative to a non-cancerous sample or a pre-determined standard valuefor a non-cancerous sample indicates the presence of cancer in thesubject. 2-4. (canceled)
 5. The method of claim 1, wherein the SGA-56Mgene product is a nucleic acid encoding SEQ ID NO:5 or SEQ ID NO:6. 6.The method of claim 1, wherein the SGA-56M gene product is a proteincomprising SEQ ID NO:5 or SEQ ID NO:6.
 7. The method of claim 1, whereinthe SGA-56M gene product is an mRNA corresponding to SEQ ID NO: 1, SEQID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
 8. The method of claim 1, whereinan antibody immunologically specific for an SGA-56M gene product is usedfor detecting or measuring the SGA-56M gene product. 9-26. (canceled)27. A method for treating a cancer in a subject, comprisingadministering to the subject a therapeutically effective amount of acompound capable of antagonizing expression and/or activity of anSGA-56M gene product, wherein said SGA-56M gene product is: (a) an RNAcorresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4;(b) a protein comprising SEQ ID NO:5; (c) a protein comprising SEQ IDNO:6; (d) a nucleic acid comprising a sequence hybridizable to SEQ IDNO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a complementthereof, or a protein comprising a sequence encoded by said hybridizablesequence; (e) a nucleic acid at least 90% homologous to SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or a complement thereof, or aprotein encoded thereby; wherein said administering reduces expressionand/or activity of the SGA-56M gene product.
 28. The method of claim 27,wherein the compound decreases expression of the SGA-56M gene product,wherein the SGA-56M gene product is a nucleic acid encoding SEQ ID NO:5or SEQ ID NO:6.
 29. The method of claim 27, wherein the compounddecreases expression of the SGA-56M gene product, wherein the SGA-56Mgene product is a protein comprising SEQ ID NO:5 or SEQ ID NO:6.
 30. Themethod of claim 27, wherein the compound decreases expression of theSGA-56M gene product and wherein the SGA-56M gene product is an RNAcorresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.31. The method of claim 27, wherein the cancer is breast or lung cancer.32-34. (canceled)
 35. The method of claim 27, wherein the compound is anantibody immunogically specific for an SGA-56M gene product. 36-37.(canceled)
 38. The method of claim 27, wherein the compound is capableof modulating expression and/or activity of a specific binding partnerof an SGA-56M molecule.
 39. The method of claim 38, wherein saidspecific binding partner is a peptide, protein, or nucleic acidsequence.
 40. The method of claim 38, wherein said SGA-56M molecule isselected from the group consisting of an SGA-56M protein or variantthereof or a nucleic acid sequence encoding an SGA-56M protein orvariant thereof. 41-99. (canceled)
 100. An immunogenic compositioncomprising: (a) an isolated SGA-56M gene product in an amount effectiveto elicit an immune response, wherein said gene product is: (i) an RNAcorresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4;(ii) an isolated protein comprising SEQ ID NO:5; (iv) an isolatednucleic acid comprising a sequence hybridizable to SEQ ID NO: 1, SEQ IDNO:2, SEQ ID NO:3, or SEQ ID NO:4, or a complement thereof, underconditions of high stringency, or a protein comprising a sequenceencoded by said hybridizable sequence; (v) an isolated nucleic acid atleast 90% homologous to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ IDNO:4, or a complement thereof, or a protein encoded thereby; and (b) apharmaceutically acceptable carrier.
 101. The immunogenic composition ofclaim 100, wherein the SGA-56M gene product is a nucleic acid at least90% homologous to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ IDNO:4.
 102. The immunogenic composition of claim 100, wherein thecomposition is an antibody immunologically specific for a proteincomprising SEQ ID NO:5 or SEQ ID NO:6. 103-110. (canceled)
 111. Theimmunogenic composition of claim 100, wherein the composition is anantibody immunologically specific for an SGA-56M molecule. 112-115.(canceled)
 116. The immunogenic composition of claim 100, wherein theSGA-56M gene product is a protein comprising SEQ ID NO:5 or SEQ ID NO:6.117. The immunogenic composition of claim 100, wherein the compositionis an antibody immunologically specific for a protein comprising SEQ IDNO:5 or SEQ ID NO:6.